1.25 Lignans: Biosynthesis and Function

Home , Enterodiol, Lariciresinol, Matairesinol, Megaphone (molecule), Pinoresinol, Secoisolariciresinol

NORMAN G. LEWIS and LAURENCE B. DAVIN Washington State University, Pullman, WA, USA

0[14[0 INTRODUCTION 539

0[14[1 DEFINITION AND NOMENCLATURE 539

0[14[2 EVOLUTION OF THE LIGNAN PATHWAY 531

0[14[3 OCCURRENCE 534 0[14[3[0 Li`nans in {{Early|| Land Plants 534 0[14[3[1 Li`nans in Gymnosperms and An`iosperms "General Features# 536

0[14[4 OPTICAL ACTIVITY OF LIGNAN SKELETAL TYPES AND LIMITATIONS TO THE FREE RADICAL RANDOM COUPLING HYPOTHESIS 536

0[14[5 707? STEREOSELECTIVE COUPLING] DIRIGENT PROTEINS AND E!CONIFERYL ALCOHOL RADICALS 541 0[14[5[0 Diriènt Proteins Stipulate Stereoselective Outcome of E!Coniferyl Alcohol Radical Couplin` in Pinoresinol Formation 541 0[14[5[1 Clonin` of the Gene Encodin` the Diriènt Protein and Recombinant Protein Expression in Heteroloòus Systems 543 0[14[5[2 Sequence Homolo`y Comparisons 543 0[14[5[3 Comparable Systems 543 0[14[5[4 Perceived Biochemical Mechanism of Action 546

0[14[6 PINORESINOL METABOLISM AND ASSOCIATED METABOLIC PROCESSES 547 0[14[6[0 Sesamum indicum] "¦#!Piperitol\ "¦#!Sesamin\ and "¦#!Sesamolinol Synthases 547 0[14[6[1 Magnolia kobus] Pinoresinol and Pinoresinol Monomethyl Ether O!Methyltransferase"s# 550 0[14[6[2 Forsythia intermedia and Forsythia suspensa 551 0[14[6[2[0 "¦#!Pinoresinol:"¦#!lariciresinol reductase 552 0[14[6[2[1 "−#!Secoisolariciresinol dehydro`enase 554 0[14[6[2[2 Matairesinol O!methyltransferase 556 0[14[6[3 Linum usitatissimum] "−#!Pinoresinol:"−#!Lariciresinol Reductase and "¦#!Secoisolariciresinol Glucosyltransferase"s# 557 0[14[6[4 Thuja plicata and Tsuga heterophylla] Pinoresinol:Lariciresinol Reductases and Other Enzymatic Conversions 557 0[14[6[5 Linum ~avum and Podophyllum hexandrum] Podophyllotoxin and its Pinoresinol Precursor 560

0[14[7 ARE DIRIGENT PROTEIN HOMOLOGUES INVOLVED IN OTHER 707? PHENOXY RADICAL COUPLING PROCESSES< 561 0[14[7[0 Li`ballinol "p!Coumarylresinol# and Related Structures 561 0[14[7[1 Syrin`aresinol and Medioresinol 562 0[14[7[2 Pellia Liverwort Li`nans 562 0[14[7[3 Li`nanamides 562 0[14[7[4 Guaiaretic Acid\ Ste`anacin\ and Gomisin A 565

0[14[8 MISCELLANEOUS COUPLING MODES] ARE DIRIGENT PROTEINS ALSO INVOLVED< 566

528 539 Li`nans] Biosynthesis and Function

0[14[09 704? AND 70O03? COUPLING OF MONOLIGNOLS AND ALLYLPHENOLS AND THEIR ASSOCIATED METABOLIC PROCESSES 567 0[14[09[0 Formation and Metabolism of 704? and 70O03? Linked Li`nans 579 0[14[09[0[0 Phenylcoumaran 60O03? rin` reduction 570 0[14[09[0[1 Allylic 607? bond reduction 571 0[14[09[1 Reìospeci_c O!Demethylation at Carbon 2? and Monosaccharide Functionalization 572 0[14[09[2 Acylation 573 0[14[00 MIXED DIMERS CONTAINING MONOLIGNOLS AND RELATED MONOMERS 574 0[14[01 LIGNANS AND SESQUILIGNANS] WHAT IS THE RELATIONSHIP TO LIGNIN FORMATION< 576 0[14[02 PHYSIOLOGICAL ROLES IN PLANTA 589 0[14[02[0 Antioxidant Properties 589 0[14[02[1 Antifunàl and Antimicrobial Effects 580 0[14[02[2 Insecticides\ Nematocides\ Antifeedants\ and Poisons 582 0[14[02[3 Allelopathy 584 0[14[02[4 Cytokinin!like Activities 584 0[14[02[5 Constitutive and Inducible "Oliòmeric# Li`nan Deposition and Nonstructural Infusions\ {{Abnormal|| and {{Stress|| Li`nins 585 0[14[03 ROLES IN HUMAN NUTRITION:HEALTH PROTECTION AND DISEASE TREATMENT 587 0[14[03[0 Nutrition:Health and Protection aàinst Onset of Breast and Prostate Cancers] Secoisolariciresinol\ Matairesinol\ and Sesamin 587 0[14[03[1 Antitumor Properties] Podophyllotoxin and other 707? Li`nans 690 0[14[03[2 Hepatotoxic Preventive Effects 691 0[14[03[3 Antiviral Properties 691 0[14[03[4 Miscellaneous Health Bene_ts] Anti!in~ammatory\ Antiasthmatic\ and Antidepressant Effects 693 0[14[04 CONCLUDING REMARKS 695 0[14[05 REFERENCES 696

0[14[0 INTRODUCTION The lignans are a very common\ structurally diverse\ group of plant natural products of phenyl! propanoid origin[0 They display important physiological functions in planta\ particularly in plant defense\1Ð4 and are most e.cacious in human nutrition and medicine\ given their extensive health protective and curative properties[5Ð8 This chapter primarily describes the intricate biochemical pathways established in lignan biosyn! thesis\ particularly as regards phenylpropanoid coupling[ It must be emphasized at the outset\ however\ that the biochemical outcome of enzymatic coupling was previously widely held to result only in formation of randomly linked racemic products[ This hypothesis\ originally adopted for lignin biopolymer formation "but see Chapter 2[07#\ could not explain the observed optical activities of the vast majority of naturally occurring lignans "discussed in Section 0[14[4#[ In contrast\ research has established that most\ if not all\ phenylpropanoid coupling reactions catalyzed by puri_ed plant proteins and enzymes in vitro can be either regio! or stereoselectively controlled\ as are their subsequent stereospeci_c metabolic conversions[ This contribution therefore attempts to summarize the progress made in this area\ as well as in providing an assessment of the rapidly growing importance of lignans in plant growth\ development\ and survival\ and in human usage[

0[14[1 DEFINITION AND NOMENCLATURE Around the turn of the nineteenth century\ a number of plant phenolic substances from various species were isolated and given trivial names\ prior to their chemical structures being determined[ One of these\ guaiaretic acid\ from guaiacum resin obtained from Guaiacum of_cinale heartwood\ was later shown to contain the skeletal formula "0# by Schroeter et al[09 It was subsequently proposed00 that it and related compounds represented a unique class of dimeric phenylpropanoid substances linked exclusively through 707? bonds\ e[g[\ "1#[ In order to provide a system for their classi_cation\ Haworth00 introduced the term {{lignane|| "later shortened to lignan# to de_ne these substances^ they were considered to result from regiospeci_cally linking two {{cinnamyl|| "C5C2# molecules to give compounds such as guaiaretic acid "0# and pinoresinols "2a\b#[ This initial classi! _cation\ unfortunately\ failed to account for either other dimeric lignan skeletal types that were Li`nans] Biosynthesis and Function 530 present in many plant species and tissues or much larger molecules "oligomeric lignans# that could also exist[ A derivative term\ neolignan\ was introduced to account for the other coupling modes\ e[g[\ megaphone "3#"700? linked#\ dehydrodiconiferyl alcohols "4a\b#"704? linked# and erythro:threo guaiacylglycerol 70O03? coniferyl alcohol ethers "5a\b#"70O03? linked#\01Ð03 but this was later modi_ed to encompass only presumed allylphenol!derived coupling products\ such as the lignans

OMe OMe

6 7 9 OH HO MeO 5 1 8 8 4 8' O O 2 8' HO 7' 3 9' 1' 8' 8 6' 2' 8 8'

3' O O 5' 4' OMe OH HO OH OMe OMe (1) Guaiaretic acid (2) (3a) (+)-Pinoresinol (3b) (Ð)-Pinoresinol [α]D = Ð94û α 22 α 25 [ ] D = +61.6û (CHCl3) [ ]D = Ð34.7û (CHCl3, c = 0.91) (Guaiacum officinale) (Forsythia europaea) (Daphne tangutica)

OH OH 4' O OH 8 O HO OH 5' MeO OH 1' MeO 8 8 OMe O OMe OMe MeO HO OH OMe OMe (4) (Ð)-Megaphone (5a,b) (±)-Dehydrodiconiferyl alcohols (6a,b) (±)-erythro/threo Guaiacylglycerol α 27 8ÐOÐ4' coniferyl alcohol ethers [ ]D = Ð23.0û (EtOH, c = 0.15) (Aniba megaphylla)

O O OH OH OH O O 8 55' 3O 4' MeO O 7' OH

71' OMe MeO O 8 O 2' O MeO OMe (7) Magnolol (8) Isomagnolol (9) Chrysophyllon 1A (10) (Magnolia virginiana) (Sassafras randaiense) (Licaria chrysophylla) (Virola sebifera)

MeO HO OH 8' O OMe 7' 7 2 O MeO 3' O 8 OMe OH MeO MeO MeO O 9 MeO OMe OMe O (11) Isoasatone (12) Lancilin (13) (Ð)-Cryptoresinol α 20 (Aniba lancifolia) α 25 [ ]D = 0û [ ]D = Ð170.4û (MeOH, c = 1.25) (Asarum taitonense) (Cryptomeria japonica) 531 Li`nans] Biosynthesis and Function

OMe H MeO 8' HO OH 8 O 8 8' 8' RO 7' MeO 7 OH OH O OMe OMe O 8 OH 9 MeO OMe OMe OH (16) (Ð)-Nortrachelogenin, R = H (14) (Ð)-Hydroxysugiresinol (15) Pachypostaudin A [α]17 = Ð16.8û (EtOH, c = 0.178) [α]25 = Ð19û (EtOH, c 1) [α]24 = 0û (CHCl , c = 0.8) D D D 3 (17) (Ð)-Trachelogenin, R = Me (Sequoia sempervirens) (Pachypodanthium staudtii) α 23 [ ] D = Ð43.3û (EtOH, c = 0.25) (Trachelospermum asiaticum)

"3# and "6#Ð"01#[04 As a further complication\ the term norlignan05 was adopted to depict lignan!like metabolites which lacked either a carbon at the C!8 and:or C!8? positions\ e[g[\ cryptoresinol "02#\06 hydroxysugiresinol "03#\07 and pachypostaudin A "04#\08 or a methyl group on the aromatic methoxyl\ e[g[\ nortrachelogenin "05#19\10 versus trachelogenin "06#[19\11 It is now well established that the products of phenylpropanoid coupling have a range of structural motifs7\03\04\12\13 and molecular sizes\14Ð29 rather than being restricted to 707? linked moieties as previously contemplated[ Moreover\ since there appear to be only a relatively small number of distinct skeletal forms\ the term lignan can be conveniently used to encompass all skeletal types\ provided that the precise linkage type is stipulated\ e[g[\ 707?\ 700?\ 704?\ 70O03?\ 404?\ 20O03?\ 600?\ 706?\ 004?\ and 10O02?[ Of the various lignan types known\ however\ those that are 707? linked appear to be the most widespread in nature\ based on current chemotaxonomic data^12 they can also be further subdivided into the substituted furofurans\ tetrahydrofurans\ dibenzylbutanes\ dibenzylbutyrolactones\ aryltetrahydronaphthalenes\ arylnaphthalenes\ dibenzocyclooctadienes\ etc[ "e[g[\ Structures "07#Ð "14##[ Although not lignans proper\ there are also related natural products of mixed metabolic origin\ e[g[\ so!called ~avonolignans20Ð22 which are described in Section 0[14[00[ Finally\ given that lignans can also exist in oligomeric form\14Ð18 it is necessary at this point to introduce terminology to distinguish them from the corresponding lignin biopolymers[ In this regard\ since upper limits of molecular size have not been established for the oligomeric lignans\ perhaps the easiest distinction lies with their physiological roles:functions] the lignin biopolymers have cell wall structural roles\ whereas oligomeric lignans are nonstructural components "discussed in Section 0[14[02[5#[

0[14[2 EVOLUTION OF THE LIGNAN PATHWAY The evolutionary adaptation of plants to land was accompanied by massive elaboration of the phenylpropanoid pathway[12 The salient features are summarized in Figure 0\ and the reader is referred to Chapter 2[07 for a comprehensive description of each enzymatic step in the pathway[ Nevertheless\ assimilated carbon is directed to provide a variety of plant phenolics\ such as the lignans and lignins\ suberins and ~avonoids\ with the latter two metabolic classes being formed via merging both phenylpropanoid and acetate pathways[ Accordingly\ the transition of plants from an aquatic to a terrestrial environment was greatly facilitated via formation of lignans "pro! tective:defense functions#\ lignins "structural support#\ suberins "formation of protective barriers preventing excessive water loss#\ and ~avonoids "~ower petal pigments\ signaling molecules\ UV!B screening\ and so forth#[12 Together\ these metabolites account for more than 29) of the carbon in vascular plants^ however\ note that lignans and lignins have never been convincingly demonstrated as present in aquatic plants\ such as algae[ Li`nans] Biosynthesis and Function 532 Furofuran Tetrahydrofuran O OMe OMe O OH HO

O O O

8' 8 8 8 8' 8' O HO OH

O HO OH O OMe OMe (18) (+)-Sesamin (19a) (+)-Lariciresinol (19b) (Ð)-Lariciresinol 20 25 25 [α]D = +68.36¡ (CHCl3, c = 24.45) [α]D = +18¡ (Me2CO, c = 1.0) [α]D = Ð12.3¡ (Me2CO, c = 0.94) (Sesamum indicum) (Araucaria angustifolia) (Daphne tangutica)

Dibenzylbutane MeO 8' 8' OMe OH HO OH HO HO 88OH

OMe MeO OH OH (20a) (Ð)-Secoisolariciresinol (20b) (+)-Secoisolariciresinol 25 [α]D = Ð35.6¡ (Me2CO, c = 1.07) (Linum usitatissimum) (Podocarpus spicatus)

Dibenzylbutyrolactone

MeO 8' 8' OMe OO

RO 8 8 OR OO

OMe MeO OH OH (21a) (Ð)-Matairesinol, R = H (21b) (+)-Matairesinol, R = H 18 20 [α]D = Ð48.6¡ (Me2CO, c = 2.409) [α]D = +37.0¡ (MeOH, c = 1.0) (Podocarpus spicatus) (Selaginella doederleinii) (22a) (Ð)-Arctigenin, R = Me (22b) (+)-Arctigenin, R = Me 23 23 [α]D = Ð34.6¡ (EtOH) [α]D = +28.05¡ (EtOH, c = 1.23) (Forsythia intermedia) (Wikstroemia indica)

Aryltetrahydronaphthalene Arylnaphthalene Dibenzocyclooctadiene OH O 8' AcO O O 8 8 O O O O O O 8 8' 8' O 2 O O 2' MeO O MeO OMe O MeO OMe OMe (23) (Ð)-Podophyllotoxin (24) Justicidin E (25) (Ð)-Steganacin [α]D = Ð109¡ (EtOH) (Justicia procumbens) [α]D = Ð122.6¡ (CHCl3, c = 1.02) (Podophyllum hexandrum) (Steganotaenia araliacea) 533 Li`nans] Biosynthesis and Function # 3!cinnamate hydroxylase\ c OH AND 2 LIGNINS CH LIGNANS** OH OH 2 Sinapyl alcohol CH (44) OH -Coumaryl alcohol OH 2 p CH ? h ? (32) OH Coniferyl alcohol (38) # tyrosine ammonia!lyase\ " b CHO OH Sinapaldehyde CHO OH (43) CHO -Coumaraldehyde SUBERIZED TISSUE* p OH Coniferaldehyde AROMATIC DOMAINS OF (31) (37) g gh # cinnamyl alcohol dehydrogenase[ ] plus acetate pathway for both ~avonoids gh h OMe OMe OMe OMe COSCoA COSCoA OH OH OMe OMe OMe OH COSCoA COSCoA COSCoA OH OH Sinapoyl CoA ester FLAVONOIDS* OH # Phenylalanine ammonia!lyase\ " Caffeoyl CoA ester HO Feruloyl CoA ester 5-Hydroxyferuloyl CoA ester a -Coumaroyl CoA ester MeO MeO MeO (42) p (34) (36) (40) (30) e e e e e H H 2 OMe H 2 OH H 2 OMe 2 H CO 2 OMe CO CO CO Caffeic acid OH CO OH OH ff OH dd Ferulic acid Sinapic acid dd -Coumaric acid OH and suberins\ ] main sources of lignan skeleta[ p (33)

ff # cinnamoyl!CoA reductase\ and " (35) (41) g 5-Hydroxyferulic acid (29) HO MeO (39) H 2 b Cinnamic acid (28) ac !methyltransferases\ " O # f 2 2 HCO H 2 2 NH NH CO CO Tyrosine Phenylalanine OH (27) (26)

# CoA ligases\ " e The phenylpropanoid pathway and selected metabolic branchpoints[ " # hydroxylases\ " d " Figure 0 Li`nans] Biosynthesis and Function 534 0[14[3 OCCURRENCE Lignans are present in a large number of vascular plant species\ ranging from {{primitive|| hornworts\ liverworts\ and ferns to the woody gymnosperms and woody:herbaceous angiosperms[ They have been found in all plant parts including "woody# stems\ rhizomes\ roots\ seeds\ oils\ exuded resins\ ~owers\ fruits\ leaves\ and bark tissues[7\12\13\23 However\ their amounts can vary extensively between tissues and species\ and in some instances their deposition can be massive[ For example\ in western red cedar "Thuja plicata# heartwood\ dimeric lignans and higher oligomers can constitute up to 19) of the dry weight of the plant tissue\24 where both "oligomeric# lignans and lignin biopolymers coexist in the same "heartwood# tissue[25 In terms of localization of lignans in planta\ evidence points to di}erent sites depending upon the tissue:species involved[ For example\ in western red cedar heartwood\ they appear to be mainly deposited as nonstructural infusions "secreted from specialized cells such as the ray parenchyma# into the preligni_ed sapwood\17 whereas in ~ax "Linum usitatissimum# they seem to be covalently bound to carbohydrate moieties in the seed[ On the other hand\ in Linum ~avum\ lignans apparently accumulate in vacuoles "discussed in Section 0[14[02[5#[26 The lignans are a structurally very diverse class of natural products\ with more than several thousand distinct structures known at the dimer level alone[7\03Ð05\27Ð31 Interestingly\ they are typically found in optically active form\ although the particular antipode can vary with plant species[ For example\ "¦#!pinoresinol "2a# is present in Forsythia europaea\32 whereas its "−#!antipode "2b# occurs in Daphne tan`utica\33 and "−#!arctigenin "11a# has been isolated from Forsythia intermedia\34 with the "¦#!form "11b# being found in Wikstroemia indica[35 A discussion of optical activity in lignans and radical coupling mechanisms is given in Section 0[14[4[

0[14[3[0 Lignans in {{Early|| Land Plants Based on DNA sequence analyses and neontological:paleontological data\36Ð38 it is considered that bryophyte!like plants\ which include the liverworts "Hepaticae#\ hornworts "Anthocerotae#\ and mosses "Musci#\ were part of the terrestrial ~ora that emerged some 19Ð39 million years or so prior to the {{pretracheophytes|| and vascular plants[ The earliest documented examples of presumed bryophyte!like fossilized remains come from both lower Devonian "½397 million years ago# and early Silurian "½327 million years ago# records] the early Silurian spore fossils possess cell wall ultrastructures reminiscent of those present in extant liverworts\49 whereas lower Devonian fossils show some similarities to modern thaloid hepatics "liverworts#[40 Moreover\ gene sequence analyses of the large subunit of ribulose 0\4!bisphosphate carboxylase:oxygenase "rbcL# in the chloroplast\ has suggested that liverworts are at the base of the embryophyte "embryo!containing plants# lineage and that the hornworts represent the closest lineage to vascular plants\41 i[e[\ the liverworts emerged _rst\ then the mosses\ and _nally the hornworts\ before the _rst tracheophytes[ Lignans are found in both liverworts and hornworts\ although none have been reported in mosses[ Consideration of their structures is of interest\ as it may give useful insight into how the lignan pathway evolved[ Thus\ lignans present in liverworts "Pellia epiphylla "34#Ð"37#\42\43 Jamesoniella autumnalis "38#Ð"43#\44 and Scapania undulata "43#45# contain optically active 707? linked lignans which provisionally appear to be ca}eic acid "22# derived[ They can also be ligated to shikimic acid moieties "44# as shown in "36#\42 or contain pendant aryl groups that have undergone _ssion and recyclization to a}ord lactones as in "38#Ð"41# and "43#[43 On the other hand\ egonol!1! methylbutanoate "45# and the optically active lignan\ "−#!licarin A "46#\ present in the liverworts\ Riccardia multi_da subsp[ decrescens46\47 and Jackiella javanica\48\59 respectively\ appear to be derived from 704? coupling of allylphenols\ eugenol "47#:isoeugenol "48# and p!hydroxyarylpropene "59#[ Of these\ egonol!1!methylbutanoate "45#inR[ multi_da has two noteworthy features] namely\ the apparent absence of a C!8 carbon and the introduction of a methylenedioxy group[ Hitherto\ methylenedioxy bridge formation was thought to occur in the lignan pathway with the advent of the gymnosperms[12 The only report of a lignan present in hornworts "Me`aceros ~a`ellaris\ Notothylas temperata\ and Phaeoceros laevis#50\51 is that of "¦#!megacerotonic acid[ Two di}erent structures "50# and "51# were reported by the same authors for this metabolite in two di}erent publications in the same year\ but without any cross!referencing or explanation given to account for the di}erent skeletal representations[50\51 In a subsequent total chemical synthesis study by Brown et al[\52 the structure of megacerotonic acid was unequivocally established to be compound "50#[ 535 Li`nans] Biosynthesis and Function

O CO H 2 HO 8 O HO 8 CO2H O CO2H HO CO H HO 8 8' 2 O OH O HO 8' CO2R O O OH HO 8' CO2H HO HO OH HO O

OH HO OH HO OH

α 20 α 20 20 ( 45) R = H, [ ]D = Ð130.77û (MeOH) (47) [ ]D = Ð122.19û (MeOH) (48) Pelliatin [α]D = Ð106.68û (MeOH) 20 (Pellia epiphylla) (46) R = Me, [α]D = Ð112.11û (MeOH) (Pellia epiphylla) (Pellia epiphylla)

O O O O HO 8 HO 8 HO 8 1 HO 8 OR OH OH OH OR2 8' OH 8' HO 8' HO HO HO 8' O O O O O O O O HO HO O HO O OH HO O 1 2 α 20 α 20 (49) R = R = H, [ ]D = +54.88û (52) [ ]D = +156.52û (53) (54) Scapaniapyrone A (MeOH, c = 0.30) (MeOH, c = 0.08) (Jamesoniella autumnalis) (Jamesoniella autumnalis) 1 2 20 (Jamesoniella autumnalis) (Scapania undulata) (50) R = Me, R = H, [α]D = +33.49û (MeOH, c = 1.70) 1 2 20 (51) R = H, R = Me, [α]D = +50.48û (MeOH, c = 0.95) (Jamesoniella autumnalis)

CO H O 2 O 8 8 5' 5' O O HO O HO OH O MeO OH OMe OMe (55) Shikimic acid (56) Egonol 2-methylbutanoate (57) (Ð)-Licarin A (Riccardia multifida, subsp. decrescens) [α]D = Ð43û (c 9.0) (Jackiella javanica)

OMe OMe OH OH OH (58) Eugenol (59) Isoeugenol (60) p-Hydroxyarylpropene Li`nans] Biosynthesis and Function 536

HO O HO O 8' 8' 8 O HO C 7 O 2

CO H HO 2 OH OH OH (61) (62) (+)-Megacerotonic acid [α]D = +233.0û (5% AcOH, c = 1.66) (Megaceros flagellaris)

Lignans are also found in the ferns "Pteridophytes#\ and include the presumed ca}eic acid "22# derived 701? linked lignans\ "−#!blechnic acid "52# and its shikimate derivative\ "−#!brainic acid "53# from Blechnum orientale\ Struthiopteris amabilis\ Struthiopteris niponica\ Woodwardia orientalis\ Woodwardia prolifera\ and Brainea insi`nis "Blechnaceae#[53 Additionally\ "−#!lirioresinol A ð"−#!epi!syringaresinolŁ "54#\ "−#!lirioresinol B ð"−#!syringaresinolŁ "55b#\ "¦#!wikstromol ð"¦#!nortrachelogeninŁ "56#\ "−#!nortracheloside "57#\ and "¦#!matairesinol "10b# are present in Sela`inella doederleinii Hieron[ "Selaginellaceae#\ a small perennial pteridophyte found in south and southwestern China at low altitude[54 The remaining lignans known to be present in the fern Pteris vittata "Pteridaceae#\ are the 704? and 707? linked glucosides of "−#!dihydrodehydrodiconiferyl alcohol "58# and "¦#!lariciresinol "69#[55 Initially\ lignan "58# was claimed to have the cis con_guration at positions 6 and 7^ however\ subsequent studies by Wallis and co!workers established it to be trans "60#[56 Other than the lignan dimers\ no higher oligomers have been reported in either liverworts\ hornworts\ or ferns[

0[14[3[1 Lignans in Gymnosperms and Angiosperms "General Features# In contrast to the relatively few examples of lignans in early land plants\ the gymnosperms were accompanied by a massive increase in lignan structures\ particularly representatives of the optically active 707? linked tetrahydrofurans\ dibenzylbutanes\ dibenzylbutyrolactones\ aryltetrahydro! naphthalenes\ and arylnaphthalenes[12 The vast majority reported thus far are E!coniferyl alcohol "27#!derived dimers^ however\ they can also exist in trimeric and higher oligomeric forms[14Ð18\25 Other lignan types\ e[g[\ 704? and 70O03? linked\ are present in varying amounts in the gymnosperms[ The evolutionary transition to the angiosperms also witnessed a massive increase in formation of "oligomeric# lignan structural types[ While the most widespread are again the optically active 707? linked\ other coupling modes\ a}ording 700?\ 704?\ 404?\ 600?\ 706?\ 004?\ 70O03?\ 10O02?\ and 20O03? coupled products also appeared*particularly in the Magnolii~orae[12 By contrast\ relatively few lignans of any type have been reported in the monocotyledons[ The few described primarily consist of cyclobutane dimers\ such as the dihydroxytruxillic acids "61# and "62# from Setaria anceps cv Nandi "Poaceae\ Commelini~orae#57\58 and acoradin "63# found in Acorus calamus "Araceae\ Ari~orae#[69 However\ rather than attempting to distinguish between the various lignans on the basis of plant family or origin\ it is perhaps more instructive to discuss the distinct structural types in terms of known or perceived biosynthetic pathways\ as understood at the enzyme\ protein\ and molecular levels[ A discussion of optical activity is\ however\ _rst required[

0[14[4 OPTICAL ACTIVITY OF LIGNAN SKELETAL TYPES AND LIMITATIONS TO THE FREE RADICAL RANDOM COUPLING HYPOTHESIS No description of lignan structure would be complete without _rst discussing the changing paradigm for control of enzymatic free!radical coupling of phenylpropanoids[ Initially\ based on the study of presumed lignin biopolymer formation\ a random free!radical coupling process was favored as being the only operative mechanism[60\61 However\ this earlier concept has been dra! matically revised\62\63 and it is important to discuss the basis for this changing view[ 537 Li`nans] Biosynthesis and Function OMe OH OH OH OH OH O O OH O OMe OH 2' 8 8' H 2' 8 O H O OH 8 CO2H MeO O CO2H OH CO2H HO HO2C OMe (63) (Ð)-Blechnic acid (64) (Ð)-Brainic acid (65) (Ð)-Lirioresinol 23 24 [α]D = Ð28¡ (MeOH, c = 1.0) [α]D = Ð42¡ (MeOH, c = 1.0) = (Ð)-Episyringaresinol (Blechnum orientale) (Blechnum orientale) (Selaginella doederleinii)

OMe OMe OH HO

O OMe MeO O

8' 8' 8 8 MeO O O OMe

HO OH OMe OMe (66a) (+)-Lirioresinol B (66b) (Ð)-Lirioresinol B = (+)-Syringaresinol = (Ð)-Syringaresinol (Selaginella doederleinii)

H H MeO MeO 8' O 8' O 8 8 HO HO OH O OH O

OMe OMe OGlc OGlc (67) (+)-Wikstromol (68) (Ð)-Nortracheloside = (+)-Nortrachelogenin (Selaginella doederleinii) (Selaginella doederleinii)

In the original random free!radical coupling hypothesis\ substrate monomers\ such as the mono! lignols\ p!coumaryl "21#\ coniferyl "27#\ and sinapyl "33# alcohols\ were envisaged to undergo single! electron oxidations "oxidase!catalyzed# to a}ord the corresponding free!radical species\ where the only enzymatic requirement was generation of the free!radical intermediates[ Under such conditions\ at least in vitro\ random coupling can then occur at several sites on the molecule to initially a}ord racemic lignan dimers\ and Figure 1 illustrates this random coupling hypothesis using E!coniferyl alcohol "27# as an example] the major coupling products formed are the three racemic 704?\ 707?\ and 70O03? linked lignan dimers "4#\ "2#\ and "5#\ with the 704? dimer predominating in coupling frequency in vitro[ According to this view\ there was believed to be no further requirement for additional enzyme: protein involvement in determining the outcome of lignin biopolymer assembly\ other than reoxi! dation of the dimeric species[ Therefore\ unlike any other biopolymer\ lignin formation was thought to be satisfactorily duplicated by the random encounter of its free!radical precursors in vitro[ However\ this random assembly mechanism\ in fact\ never gave a biopolymer duplicating lignin structure "see Chapter 2[07#^ nor could it explain the observed optical activity of numerous lignans Li`nans] Biosynthesis and Function 538 OMe OGlc OH

8 O 5' OH HO 8' 8 7 O 4' HO MeO OMe GlcO MeO

(69) cis-Dihydrodehydrodiconiferyl alcohol-9ÐOÐβ-D-glucoside (70) Lariciresinol-9ÐOÐβ-D-glucoside (incorrectly assigned) 27 [α]D = Ð39.77¡ (Me2CO, c = 0.88) 20 [α]D of aglycone = +15.7¡ (Me2CO, c 0.35) OGlc (Pteris vittata)

8 5' OH HO 7 O 4' MeO OMe

(71) trans-Dihydrodehydrodiconiferyl alcohol-9ÐOÐβ-D-glucoside 22 [α]D = Ð23.6¡ (MeOH, c = 0.98) 22 [α]D of aglycone = +8.5¡ (Me2CO, c = 0.96) (Pteris vittata)

OH R OH OH R OMe 88' OMe CO2H R 8 8' 8 HO2C MeO OMe CO2H 8' OMe OMe CO H R 2 OH (72) Dihydroxytruxillic acid (73) Dihydroxytruxinic acid (74) Acoradin R = H or OMe R = H or OMe [α]D = 0¡ (c = 1.1) (Setaria anceps) (Setaria anceps) (Acorus calamus)

occurring in a wide variety of plant species[ Indeed\ lignan optical activity could presumably only result via one of three ways] "i# where stereoselective phenylpropanoid coupling occurs\ thereby explicitly controlling both the regio! and stereochemistries^ "ii# where racemic coupling occurs\ but where one of the enantiomeric forms is selectively metabolized^ and:or "iii# where both stereoselective and random coupling can occur\ but to di}erent extents in di}erent tissues and cell types[ Each of these three possibilities had to be considered since\ in many cases\ the optical rotation\

ðaŁD\ value for a particular lignan can di}er substantially between plant species] For example\ Table 0 shows the variation in reported optical rotations for syringaresinols "55# obtained from di}erent plants[33\64Ð74 As can be seen\ these range from being optically active ð"¦#! or "−#!Ł\ to being nearly racemic[ These observations\ however\ only serve to remind us that an ðaŁD determination does not reveal the enantiomeric purity of isolated lignans\ or\ indeed\ provide any insight as to whether the enantiomer"s# originate from the same subcellular compartment\ cell type\ or tissue[ The application of chiral HPLC methodologies\ suitable for the facile separation of various lignan optical antipodes\ has signi_cantly helped in the study of this class of natural products\ i[e[\ whether for examination of enantiomeric purity and:or outcome of phenylpropanoid coupling[75Ð77 The two major chiral stationary phases currently employed for separation of "¦#! and "−#!lignan antipodes are either cellulose carbamate coated on silica gel "Chiralcel columns\ Daicel#\ or a macrocyclic 549 Li`nans] Biosynthesis and Function 8ÐOÐ4' linked 8Ð8' linked 8Ð5' linked (6a,b) (±)-Erythro/threo guaiacyl glycerol (3a,b) (±)-Pinoresinols (5a,b) (±)-Dehydrodiconiferyl alcohols 8ÐOÐ4' coniferyl alcohol ethers

HO OH OH O OH HO O OMe OH OMe HO C O O E HO OMe MeO L OMe L OH MeO

W A Intramolecular cyclization L H2O L OH

MeO HO OMe OH

OH OH ¥ OH ¥ O O ¥ ¥ ¥ ¥ 8ÐOÐ4' OMe coupling 8Ð8' 8Ð5' 1 electron coupling O oxidation coupling OMe OMe OMe O O O Plasma membrane MeO MeO OH OH Cytoplasm HO HO (38) Coniferyl alcohol (38) Coniferyl alcohol Figure 1 Illustration of the monolignol random coupling hypothesis leading to lignin as according to Freudenberg[60\61

Table 0 Speci_c rotations and enantiomeric compositions of syringaresinol "55# isolated from di}erent species[65 "Enantiomeric composition was determined following chiral column chro! matography[# *ÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐ Syrin`aresinol ")# *ÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐ

Species ðaŁD reported Ref[ "¦#!"55a# "−#!"55b# *ÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐ Aspidosperma marc`ravianum −23[7 66 Holocantha emoryi −21[4 67 Xanthoxylum ailanthoides −8[5 68 27 51 Daphne tan`utica −1[0 33 37 41 Xanthoxylum inerme 9793741 Stellera chamaejasme ¦2[9 70 Liriodendron tulipifera ¦08[9 71 Hedyotis lawsoniae ¦12[9 72 Eucommia ulmoides ¦33[9 73 77 01 Liriodendron tulipifera ¦37[8 74 *ÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐ

glycopeptide covalently bound to silica "4 mm#\ such as Vancomycin "Chirobiotic V column\ Advanced Separation Technologies#[ The basis for their separations is the reversible formation of transient diastereomeric complexes formed between each enantiomer and the chiral stationary phase[65 Li`nans] Biosynthesis and Function 540 Conditions have thus been developed for separation of "¦#! and "−#!enantiomers of various lignans obtained\ for example\ by chemical syntheses\ such as the pinoresinols "2a\b#\ seco! isolariciresinols "19a\b#\ syringaresinols "55a\b#\ and dehydrodiconiferyl alcohols "4a\b# "see Figures 2"a# to 2"d##[ Further\ chiral HPLC analysis of certain lignans from selected plant species\ e[g[\ F[ intermedia\ has revealed that\ in many instances\ lignans\ such as pinoresinol "2# and seco! isolariciresinol "19#\ occur in optically pure form "Figures 2"e# and 2"f##[ On the other hand\ with the syringaresinols "55a\b# isolated from di}erent sources\ it was observed "Figure 3 and Table 0# that both antipodes could either occur in enantiomeric excess "e[g[\ in Xanthoxylum ailanthoides and Eucommia ulmoides^ Figures 3"b# and 3"a## or were in nearly racemic amount "e[g[\ D[ tan`utica and Xanthoxylum inerme^ Figures 3"c# and 3"d##[ Some convincing scienti_c explanation was thus required for the noted\ but often di}ering\ optical activities in the lignans[

Figure 2 Chiral HPLC separation of selected lignans[ "a#"2#!Pinoresinols "2a\b#\ "b#"2#!secoisolariciresinols "19a\b#\ "c#"2#!syringaresinols "55a\b#\ "d#"2#!dehydrodiconiferyl alcohols "4a\b# as well as "e# "¦#!pinoresinol "2a#\ and "f# "−#!secoisolariciresinol "19a# isolated from Forsythia intermedia["2#!Pinoresinols "2a\b#\ "2#! syringaresinols "55a\b#\ and "2#!secoisolariciresinols "19a\b# were individually resolved using a Chiralcel OD column "Daicel#\75\77 whereas resolution of "2#!dehydrodiconiferyl alcohols "4a\b# employed a Chirobiotic V column "Advanced Separation Technologies# with MeOH!NH3NO2 "0]8# as solvent system "~ow rate] 0 ml min−0#[ 541 Li`nans] Biosynthesis and Function

Figure 3 Chiral HPLC separation of syringaresinols "55a\b# isolated from di}erent plant species[ "a# Eucommia ulmoides\"b#Xanthoxylum ailanthoides\"c#Daphne tan`utica\ and "d# Xanthoxylum inerme[65 For elution conditions\ see Figure 2 legend " contaminant#[

0[14[5 707? STEREOSELECTIVE COUPLING] DIRIGENT PROTEINS AND E!CONIFERYL ALCOHOL RADICALS

0[14[5[0 Dirigent Proteins Stipulate Stereoselective Outcome of E!Coniferyl Alcohol Radical Coupling in Pinoresinol Formation Since lignan optical activity cannot readily be explained by random coupling\ it was essential to delineate how monolignol coupling control might be achieved in planta[ Initial investigations used Forsythia species\ given that it is an abundant source of the optically pure 707? linked lignans\ "¦#! pinoresinol "2a# and "−#!matairesinol "10a#] it was discovered that Forsythia stem residues\ following removal of readily soluble proteins\ were capable of preferentially stereoselectively converting E! coniferyl alcohol "27# into the 707? linked "¦#!pinoresinol "2a#in½59) enantiomeric excess[75 In contrast\ all previous bimolecular phenoxy radical coupling processes\ whether engendered biochemically78 or chemically\60 only a}orded the racemic lignans\ "2#!dehydrodiconiferyl alcohols "4a\b#\ "2#!pinoresinols "2a\b#\ and "2#!erythro:threo guaiacylglycerol 70O03? coniferyl alcohol ethers "5a\b# as previously discussed[ "¦#!Pinoresinol "2a# was ultimately demonstrated to be formed in vitro when two distinct proteins were added together^ this resulted in conferring the requisite stereoselectivity to the coupling of two E!coniferyl alcohol "27# molecules[ The _rst protein was of circa 67 kDa molecular size\ as deter! mined by both gel permeation chromatography "Sepharose S199\ Pharmacia# and analytical ultra! centrifugation[ SDS!PAGE analysis\ on the other hand\ gave a single band of 15Ð16 kDa suggesting that the native protein existed as a trimer[ This 67 kDa protein lacked any "oxidative# catalytic capacity by itself\ and was unable to directly engender formation of "¦#!pinoresinol "2a# from E! coniferyl alcohol "27#[ The second protein exhibited a typical plant laccase EPR spectrum\ but did not catalyze stereoselective coupling[ It did\ however\ oxidatively convert E!coniferyl alcohol "27# into the well!known racemic lignan products in vitro\ i[e[ "4a\b#\ "2a\b#\ and "5a\b# in a ratio of circa 3]1]0 "Figure 4"a##[ Li`nans] Biosynthesis and Function 542

Figure 4 Time courses for E!coniferyl alcohol "27# depletion and formation of corresponding lignans during 76 incubation in the presence of "a# an oxidase\ and "b# dirigent protein and an oxidase[ Â\ Coniferyl alcohol "calculated as dimer equivalents#^ Á\"2#!dehydrodiconiferyl alcohols "4a\b#^ ž\ "¦#!pinoresinol "2a#^ R\"2#!pinoresinols "2a\b#^ r\"2#!erythro:threo guaiacylglycerol 70O03? coniferyl alcohols "5a\b#^ and 76 e\ total of all lignans "after Lewis et al[ #[

When both proteins were combined together in judicious amounts\ however\ essentially only conversion of E!coniferyl alcohol "27# into "¦#!pinoresinol "2a# was observed[ This is illustrated in Figure 4"b# which shows the e}ect of both proteins on stereoselective coupling\ and the term dirigent protein76 "Latin\ diri`ere] to guide or align# was introduced to describe the perceived unique function of the 67 kDa protein "discussed in Section 0[14[5[4#[ Signi_cantly\ stereoselective coupling occurred regardless of whether a one!electron oxidase "e[g[\ laccase# or a one!electron oxidant "such as FMN# was used in conjunction with the dirigent protein "see Figures 5"b# and 5"d##[ In both cases\ dirigent

Figure 5 Time courses for E!coniferyl alcohol "27# depletion and formation of corresponding lignans during incubation in the presence of "a# laccase\ "b# dirigent protein and laccase\ "c# FMN\ and "d# FMN and dirigent protein[ See Figure 4 for key "after Lewis et al[76#[ 543 Li`nans] Biosynthesis and Function protein addition had little\ if any\ e}ect on the rate of E!coniferyl alcohol "27# depletion "see Figures 5"a# to "d##[ Moreover\ stereoselectivity was only observed with E!coniferyl alcohol "27#asa substrate\ but not when either E!p!coumaryl "21#orE!sinapyl "33# alcohol was used[

0[14[5[1 Cloning of the Gene Encoding the Dirigent Protein and Recombinant Protein Expression in Heterologous Systems The cDNA of two Forsythia dirigent protein genes "psd!Fi0 and psd!Fi1^ Figures 6"a# and 6"b## were obtained by a polymerase chain reaction guided strategy\ and sequence analyses revealed the presence of secretory signal peptides\ potential N!glycosylation and serine:tyrosine phosphorylation sites[63\89 Each cDNA encoded a protein subunit of only circa 07 kDa\ after taking into account the secretory signaling sequences\ indicating that the presumed 15 kDa subunit was extensively glycosylated[ The authenticity of the dirigent protein clone"s# was proven by obtaining recombinant dirigent proteins] these were heterologously expressed in both Spodoptera fru`iperda "fall army worm# Sf8 cells63 using a baculovirus based expression system\80 and in Drosophila melano`aster cells cotransfected with the DES "drosophila expression system# vector "Invitrogen#^81 both expression systems were chosen over Escherichia coli since they carry out glycosylation:post!translational modi_cations[ As illustrated in Figure 7\ the puri_ed recombinant proteins were functionally capable of conferring the requisite stereoselectivity to ð8!2HŁconiferyl alcohol "27# coupling\ provided that single!electron oxidative capacity "e[g[\ laccase# was supplied\ thereby establishing the gene 63\89 1 sequence"s# to be correct[ These _ndings were also con_rmed using deuterated ð8! H1\ 1 OC H2Łconiferyl alcohol "27# as substrate[

0[14[5[2 Sequence Homology Comparisons The Forsythia dirigent protein sequence has no signi_cant level of homology to any other protein of known function when using the BLAST:BLAST!Beauty database search tool[82\83 It did\ however\ show signi_cant identity "½53)# with a {{pea disease resistance response gene 195!d|| of unknown function\ which is induced in conjunction with iso~avone reductase[84 Low sequence homology levels were also noted with portions of three other plant genes\ of no known function\ i[e[\ Arabidopsis thaliana\ wheat "Triticum aestivum#\ and barley "Hordeum vul`are#[ The Arabidopsis gene possessing limited sequence homology was the {{hypothetical protein|| random BAC clone "39) similarity over 71) of the sequence\ Genbank accession number SF999546#\ whereas that from wheat "36) similarity over 45) of the sequence\ Genbank accession number U21316# is associated with systemic acquired resistance and is induced by benzothiadiazole[85 Finally\ the barley gene encoded a {{puta! tive 21[6 kDa jasmonate!induced|| gene "Genbank accession number U32386#\ with some 49) similarity over 24) of the sequence of the dirigent protein[ No meaningful comparisons could be made with other gene sequences outside of the plant kingdom\ and it can\ therefore\ be concluded that the dirigent protein genes are unique[ Current information does not\ however\ give any incisive insight into how these gene"s# may have evolved\ or as to what its progenitor function may have been[

0[14[5[3 Comparable Systems Since various 707? linked lignans are present in many plant species\ isolation and characterization of dirigent protein analogues and homologues was of interest[ Accordingly\ dirigent protein involve!

000000000000000000000000000000000000000000000004 Figure 6 Complete sequence of Forsythia intermedia dirigent protein cDNA "a# psd!Fi0\ and "b# psd!Fi1[ The signal peptide cleavage sites are indicated by an arrow "before Arg!14 and His!14 in psd!Fi0 and psd!Fi1\ respectively\ in the native proteins\ and before Thr!11 in the recombinant psd!Fi0 proteins#[ Potential N! glycosylation sites "Asn!41\ Asn!54\ Asn!011\ and Asn!039 in psd!Fi0^ Asn!40\ Asn!53\ Asn!010\ and Asn!028 in psd!Fi1# and serine "Ser!012 in psd!Fi0\ Ser!17 in psd!Fi1# and tyrosine phosphorylation "Tyr!072 in psd! Fi0\ Tyr!071 in psd!Fi1# sites are indicated by underlining[ The stop codons are indicated by an asterisk "after Lewis et al[63#[ Li`nans] Biosynthesis and Function 544 545 Li`nans] Biosynthesis and Function

Figure 7 Chiral HPLC analysis of ð8\8?!2HŁ!pinoresinol "2# formed by incubation of recombinant dirigent protein with ð8!2HŁconiferyl alcohol "27# in the presence of laccase as oxidant[63 For elution conditions and column employed see Figure 2 legend[

ment in "¦#!pinoresinol formation was detected in sesame "Sesamum indicum#\ whereas in ~ax "Linum usitatissimum# the corresponding dirigent protein stipulated formation of the "−#!antipode86 i[e[\ the proteins from di}erent plant sources can have di}erent stereoselectivities to give "¦#! and "−#!pinoresinols "2a# and "2b#\ respectively[ The Forsythia dirigent protein cDNA\ psd!Fi0\ was used to probe the cDNA libraries of two gymnosperms\ western red cedar "T[ plicata# and western hemlock "Tsu`a heterophylla#\ which accumulate 707? linked lignans\ plicatic acid "64# and "a#!conidendrin "65#\29 respectively\ in their heartwood[ Two dirigent protein!like cDNAs were isolated from T[ plicata "psd!Th0 and psd!Th1# and eight from T[ heterophylla "psd!Tp0 to psd!Tp7#[63\89 Using a PCR!guided approach on DNA extracted from di}erent plant species\ dirigent protein genes were also identi_ed in Manchurian ash "Fraxinus mandschurica\ Salicaceae] psd!Fm0 and psd!Fm1# and aspen "Populus tremuloides\ Oleaceae] psd!Pop0 and psd!Pop1#[ These have a very high degree of similarity:identity63 "from 80[8) and 74[3) for psd!Fm0 and psd!Fm1 versus psd!Fi0 to 55[0) and 50[1) for psd!Tp0 versus psd!Fi0#\ as shown in Table 1[

Table 1 Similarity:identity between the di}erent dirigent protein!like clones iso! lated and the dirigent protein clone psd!Fi0 from Forsythia intermedia[ *ÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐ With si`nal peptide *ÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐ Plant species cDNA Similarity ")# Identity ")# *ÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐ Forsythia intermedia psd!Fi1 76[5 70[5 Thuja plicata psd!Tp0 55[0 50[1 psd!Tp1 66[3 50[7 psd!Tp2 67[4 50[7 psd!Tp3 72[2 56[3 psd!Tp4 67[3 59[9 psd!Tp5 65[2 46[4 psd!Tp6 68[9 48[0 psd!Tp7 68[9 48[0 Tsu`a heterophylla psd!Th0 58[8 59[7 psd!Th1 57[4 50[3 Fraxinus mandschurica psd!Fm0 80[8 74[3 psd!Fm1 80[8 74[3 Populus tremuloides psd!Pop0 66[1 55[6 psd!Pop1 66[1 55[6 *ÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐ

The high similarity:identity between di}erent dirigent protein clones isolated and detected indi! cates that the dirigent proteins may be ubiquitous throughout the plant kingdom\ although it Li`nans] Biosynthesis and Function 546

OH OH O MeO MeO O CO2H HO OH HO

MeO OH OMe OH OH (75) Plicatic acid (76) (Ð)-α-Conidendrin remains to be established whether they stipulate only 707? coupling or other modes "e[g[\ 700?\ etc[# as well "see Sections 0[14[7 and 0[14[8#[

0[14[5[4 Perceived Biochemical Mechanism of Action Free!radical coupling reactions\ as catalyzed by nonspeci_c "per#oxidases\ cannot control either the regio! or stereochemistries of product formation\ when more than one potential coupling site is on the substrate molecule[ On the other hand\ nature extensively utilizes free!radical coupling processes\ with circa 29Ð39) of all organic carbon being linked together in this way\ e[g[\ lignans\ lignins\ suberins\ melanins\ insect cuticles\ etc[ Given the extensive deployment of such coupling reactions in vivo\ it could therefore be anticipated that nature had some means to control or stipulate the outcome of phenoxy free!radical coupling[ The discovery of the Forsythia dirigent protein\ stipulating both the regio! and stereochemical fate of E!coniferyl alcohol "27# coupling during "¦#!pinoresinol "2a# formation\ thus gave a new perspective as to how control of free!radical reactions occurred in vivo^ this was further supported by analysis of its unique 07 kDa gene sequence which revealed no counterpart elsewhere[ It is believed that the dirigent protein e}ectuating "¦#!pinoresinol "2a# formation requires glycosylation of the 07 kDa subunit to give the 15 kDa glycoprotein which then forms the functional ½67 kDa dirigent protein trimer[ There are three distinct biochemical mechanisms76 that can be envisaged as operative\ with each discussed in terms of relative likelihood based on data interpretation[ The _rst is generation of free! radicals from E!coniferyl alcohol "27#\ by action of nonspeci_c oxidase"s#:oxidant"s#\ with the free radicals then binding to the dirigent protein prior to stereoselective coupling[ In this case\ the free! radical species are orientated on the 67 kDa protein prior to coupling\ hence the term dirigent "Latin\ diri`ere] to guide or align#[ Alternative possibilities are that E!coniferyl alcohol "27# molecules are themselves bound to the dirigent protein\ and appropriately orientated to give "¦#!pinoresinol "2a# following one!electron oxidation[ It is anticipated that this could only occur when either the substrate phenolic groups were exposed so that they could readily be oxidized by an oxidase or oxidant\ or via an electron transfer mechanism between the oxidase:oxidant and an electron acceptor site or sites on the dirigent protein[ Lines of evidence\ however\ suggest {{capture|| of phenoxy radical intermediates by the dirigent protein[ This is because both the rates of substrate depletion and product formation were largely una}ected by the presence of the dirigent protein[ This is consistent with a free!radical capture mechanism which would only a}ect coupling speci_city when single!electron oxidation of coniferyl alcohol "27# is rate!determining[ The electron transfer mechanism is ruled out\ on the basis that no new ultraviolet!visible chromophores were observed in either the presence or absence of an auxiliary oxidase or oxidant\ under oxidizing conditions[ Preliminary kinetic data were also in agreement with free!radical capture based on the formal Michaelis constant "Km# and maximum velocity "Vmax# values characterizing the conversion of E!coniferyl alcohol "27# into "¦#!pinoresinol "2a#\ with the dirigent protein alone and in the presence of the various oxidases or oxidants[76 With a free!radical capture process\ the MichaelisÐMenten parameters obtained will only represent formal rather than true values\ given that the highest free!energy intermediate state during the conversion of E!coniferyl alcohol "27# into "¦#!pinoresinol "2a# is unknown and the relation between the concentration of substrate and that of the corresponding intermediate free!radical in open solution has not been delineated[ 547 Li`nans] Biosynthesis and Function

With these quali_cations in mind\ formal Km and Vmax values for the dirigent protein preparation were estimated in the presence and absence of the oxidase\ laccase and the oxidant\ FMN[ The preliminary kinetic parameters so obtained were in harmony with the _nding that the dirigent protein does not substantially a}ect the rate of E!coniferyl alcohol "27# depletion in the presence of the oxidase:oxidant\ and are thus in accord with the working hypothesis that the dirigent protein functions by capturing free!radical intermediates which then undergo stereoselective coupling[ Accordingly\ it is of considerable importance to establish the site"s# on the dirigent protein that involve substrate binding\ together with the number of monomeric forms binding to the 07 kDa subunit and to the preformed 67 kDa protein[

0[14[6 PINORESINOL METABOLISM AND ASSOCIATED METABOLIC PROCESSES The 707? linked lignan\ pinoresinol "2#\ is a central intermediate in lignan metabolism\ which\ depending upon the plant species\ can be converted into a rather large number of natural products of important plant physiological and pharmacological functions "see Sections 0[14[02 and 0[14[03#[ Delineation of the various biochemical pathways associated with its metabolism has been carried out using sesame "S[ indicum#\ Ma`nolia kobus\ Forsythia species\ ~ax "L[ usitatissimum#\ western red cedar "T[ plicata#\ western hemlock "T[ heterophylla#\ Linum ~avum\ and Podophyllum species[ Together\ these plants contain various furanofuran\ furano\ dibenzylbutane\ dibenzylbutyrolactone\ and aryltetrahydronaphthalene lignans\ and\ therefore\ provide the opportunity to study the for! mation of distinct 707? linked lignan skeletal forms[

0[14[6[0 Sesamum indicum] "¦#!Piperitol\ "¦#!Sesamin\ and "¦#!Sesamolinol Synthases Sesame "S[ indicum# seeds are rich in furanofuran lignans\ of which "¦#!sesamin "07# and the unusual oxygenated derivative\ "¦#!sesamolin "66# are the most abundant "Figure 8#[ They di}er from "¦#!pinoresinol "2a# by methylenedioxy bridge formation and\ in the latter case\ by an oxygen insertion between the furanofuran group and the aryl moiety[ Their physiological roles appear to be as protective antioxidants\ thereby helping stabilize sesame seed oil from the rapid onset of rancidity[3\87\88 Sesame seed pod development is a gradual maturation process\ being initiated some 03 days or so after ~owering in the oldest tissues nearest the stem base[ New seed pods are then continually formed over a six!week period\ and Figure 09 shows a maturing S[ indicum plant with its seed pods at di}erent maturation stages[ Plants at this stage were used for tracer experiments\ employing both radio! and stable isotopically labeled lignan precursors\ where it was found that sesamin "07# and sesamolin "66# were both formed de novo in maturing seed[099\090 The possible biochemical routes to both lignans are shown in Figure 8[ 03 When racemic "2#!ð2\2?!O CH2Łpinoresinols "2a\b# were administered to intact sesame seeds taken from pods from eight!week!old plants\ it was found that only the "¦#!antipode "2a# was metabolized into "¦#!piperitol "67#\ "¦#!sesamin "07#\ and "¦#!sesamolin "66#^ the corresponding "−#!enantiomer "2b# was not utilized[090 It was also found that the relative e.cacy of metabolism 03 of "¦#!ð2\2?!O CH2Ł pinoresinol "2a# into each of these lignans varied with the stage of maturation of the developing seed pod[ Incorporation into sesamolin "66# was highest at the early stages of seed pod maturation\ whereas conversion into sesamin "07# and piperitol "67# was greatest at the later stages[ 1 Con_rmation of these radiochemical observations was attained by administering "2#!ð8\8?! H1\ 1 1 2\2?!OC H2Łpinoresinols "2a\b# to sesame seed[ The enzymatically synthesized "¦#!ð8\8?! H1\ 1 1 ¦ 2!OC H1\ 2?!OC H2Łpiperitol "67# so obtained displayed a molecular ion ðM ¦8Ł at m:z 254\ corresponding to the presence of nine deuterium atoms[090 This established intact incorporation of the precursor during methylenedioxy bridge formation\ in a conversion catalyzed by "¦#!piperitol synthase[ Microsomal preparations from this seed pod maturation stage were examined as a potential 03 source of "¦#!piperitol synthase[ These were incubated with "2#!ð2\2?!O CH2Łpinoresinols "2a\b# 03 03 090 but only gave radiolabeled ð2!O CH1\ 2?!O CH2Łpiperitol "67# when NADPH "0 mM# was added[ The conversion was completely enantiospeci_c\ since only the radiolabeled "¦#!antipode was formed "Figure 00#[ Con_rmation of the radiochemical _ndings was achieved using isotopically labeled Li`nans] Biosynthesis and Function 548

OH MeO OMe OMe O OH OH O

O O O O O

O O O O HO O O HO OMe O O OMe (79) (+)-Pinoresinolin (3a) (+)-Pinoresinol (78a) (+)-Piperitol (18) (+)-Sesamin

OH MeO

O O

O O O O O

O O (81) (+)-Sesamolinol O O O O

O O

HO O OMe O (80) (+)-Piperitolin (77) (+)-Sesamolin Figure 8 Possible biosynthetic pathways to "¦#!sesamolin "66# from "¦#!pinoresinol "2a#inS[ indicum["67b would be the opposite enantiomer[#

1 1 "2#!ð8\8?! H1\ 2\2?!OC H2Łpinoresinols "2a\b# as substrates\ where the enzymatically generated "¦#!piperitol "67# displayed the expected molecular ion ðM¦ ¦8Ł at m:z 254 as before[090

"¦#!Piperitol synthase is an O1!requiring\ NADPH!dependent\ cytochrome P349 enzyme\ with temperature and pH maxima of 39 >C and 6[4\ respectively[ Cytochrome P349 inhibitors\ clo! trimeizol "299 mM#\ miconazol "299 mM#\ and cytochrome c "069 mM#\ completely inhibited the enzyme\ with tropolone and metyrapone being less e.cient "Table 2#[ It is also strongly inhibited 090 by carbon monoxide "89) inhibition in a CO:O1 "8]0# atmosphere#[ These results are consistent with previous studies of benzophenanthridine alkaloid091\092 and iso~avonoid093 biosynthetic path! ways\ where methylenedioxy bridge generation during the formation of such natural products was catalyzed by NADPH!dependent\ cytochrome P349 monooxygenase"s#[ Interestingly\ the S[ indicum microsomal preparation only e}ectively converted "¦#!pinoresinol "2a# into "¦#!piperitol "67#\ but not into "¦#!sesamin "07#[ Incubation with "¦#!piperitol "67# did\ however\ give "¦#!sesamin "07#[094 Accordingly\ there may be two distinct cytochrome P349s involved in "¦#!piperitol "67# and "¦#!sesamin "07# formation\ respectively[ The enzymology associ! ated with oxygen insertion between the aryl group and furanofuran to give sesamolin "66# also awaits delineation[ It is perhaps signi_cant\ however\ that ketone "71# was isolated from S[ indicum seeds\095 and studies will establish if it is a pathway intermediate to "¦#!sesamolin "66# via rearrange! ment[ 559 Li`nans] Biosynthesis and Function

Figure 09 Sesame "S[ indicum# plant showing pods at di}erent maturation stages[

03 03 Figure 00 Chiral HPLC separations of "¦#! and "−#!piperitols "67a\b#] "a# "¦#!ð2!O CH1\ 2?!O CH2Ł! 03 Piperitol "67a# obtained after incubation of "2#!ð2\2?!O CH2Łpinoresinols "2a\b# with a S[ indicum microsomal preparation and "b# "¦#! and "−#!antipodes of synthetic piperitols "67a# and "67b# "after Jiao et al[090#[ Li`nans] Biosynthesis and Function 550

Table 2 E}ect of various cytochrome P349 enzyme inhibitors on the activity of microsomal bound "¦#!piperitol synthase from S[ indicum[ "Standard assay conditions were employed\ where 099) activity 0[35 pkat for "¦#!piperitol synthase[#090 *ÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐ Cytochrome P349 Inhibitor Inhibition inhibitor concentration "mM# ")# *ÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐ Clotrimeizol 299 099[9 24 099[9 Miconazol 299 099[9 24 60[1 Cytochrome c 069 099[9 06 41[3 Tropolone 299 54[4 24 43[3 Metyrapone 299 35[3 24 15[7 *ÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐ

O O

HO O

O O (82) Ketone

0[14[6[1 Magnolia kobus] Pinoresinol and Pinoresinol Monomethyl Ether O!Methyltransferase"s# Ma`nolia kobus var[ borealis is a member of the Magnoliaceae\ which accumulates furanofuran and tetrahydrofuran lignans in its leaves\ e[g[\ "¦#!eudesmin "72#\ "¦#!magnolin "73#\ "¦#!yan! gambin "74#\ "¦#!kobusin "75#\ and "¦#!kobusinols A "76#andB"77#[096 Its lignans primarily di}er from pinoresinol "2# by the degree of methoxylation and:or methylenedioxy bridge formation on the aromatic rings\ as well as reductive modi_cations "discussed in Section 0[14[6[2[0#[ Thus\ the methylation patterns "i[e[\ at C!3:C!3?# for these lignans have a very di}erent regiospeci_city to that observed for monolignol formation\ which only occurs at the meta "C!2# position "see Figure 0 for comparison#[ Miyauchi and Ozawa have carried out studies to begin to characterize the M[ kobus lignan O!methyltransferases using crude cell!free extracts from leaves "Scheme 0#[097 These were individually incubated with both racemic "2#!pinoresinols "2a\b# and "2#!pinoresinol monomethyl ethers "78#\ in the presence of S!adenosyl!L!ðmethyl!03CŁmethionine\ in order to ascertain whether the O!methyltransferases were enantiospeci_c or not[ With "2#!pinoresinols "2a\b# as substrates\ 03 both "¦#! and "−#!ð3!O CH2Łpinoresinol monomethyl ethers "78# were formed\ where the "¦#! enantiomer predominated "2]0# over the "−#!form^ dimethylation a}ording "¦#!eudesmin "72#\ however\ was not observed[ On the other hand\ with "2#!pinoresinol monomethyl ethers "78#as substrates\ both "¦#! and "−#!eudesmins "72# were obtained with formation of the "¦#!enantiomer again being favored "½1]0#[ Thus\ the crude O!methyltransferase preparation catalyzing formation of eudesmin "72# in vitro from pinoresinol "2# is either not stereospeci_c\ or more likely contains various O!methyltransferases which di}er in their speci_cities towards each enantiomeric form[ These possibilities will only be distinguished when the puri_ed O!methyltransferases are obtained[ As discussed below "in Section 0[14[6[2[2#\ this lack of being fully enantiospeci_c is consistent with previous observations made\ using Forsythia crude cell!free preparations\ in the study of O!methylation of matairesinol "10#to give arctigenin "11#[098 551 Li`nans] Biosynthesis and Function

OMe OMe OMe OMe 4' O R1 O 8' 8' 8 8 R2 O O

MeO 4 O OMe O 1 2 25 25 (83) R = R = H, Eudesmin, [α]D = +64.0¡ (MeOH, c = 0.01) (86) Kobusin, [α]D = +48.0¡ (MeOH, c = 0.31) 1 2 25 (84) R = H, R = OMe, Magnolin, [α]D = +55.9¡ (MeOH, c = 0.35) 1 2 25 (85) R = R = OMe, Yangambin, [α]D = +63.49¡ (MeOH, c = 0.03)

OMe OMe OMe OMe HO HO HOH2C 88' 88'

O O

HO MeO OMe OMe 25 25 (87) Kobusinol A, [α]D = +98.2¡ (MeOH, c = 0.28) (88) Kobusinol B, [α]D = +16.4¡ (MeOH, c = 0.22)

OMe OMe OMe OH OMe OMe

O O O SAM SAM

O O O

HO HO MeO OMe OMe OMe (3) Pinoresinol (89) Pinoresinol monomethyl ether (83) Eudesmin

Scheme 1

0[14[6[2 Forsythia intermedia and Forsythia suspensa Forsythia species accumulate various 707? linked lignans in di}ering amounts] "¦#!pinoresinol "2a#\ "¦#!phillygenin "89#\ and "¦#!phyllirin "80# are present in F[ suspensa\ whereas F[ viridissima accumulates "−#!matairesinol "10a#\ "−#!arctigenin "11a#\ and "−#!arctiin "81#[ The hybrid\ F[ intermedia "F[ suspensa × F[ viridissima#\ on the other hand\ contains all of these lignans[34 The lignan biosynthetic pathway de_ned in the Forsythia plant family is summarized in Scheme 1\ being initiated with the dirigent protein mediated stereoselective coupling of E!coniferyl alcohol "27# to give "¦#!pinoresinol "2a# "Section 0[14[5#[ This is sequentially reduced to a}ord "¦#!lariciresinol "08a# and "−#!secoisolariciresinol "19a#\ respectively\ with stereospeci_c dehydro! genation of the latter giving "−#!matairesinol "10a#\ which is subsequently converted into "−#!arctigenin "11a# and "−#!arctiin "81#[ The enzymology associated with "¦#!pinoresinol "2a# metabolism in F[ intermedia is described below[ Li`nans] Biosynthesis and Function 552 OMe

OR MeO 8' O O R1O 8 H O H O OMe 2 MeO OR OMe (90) R = H, (+)-Phillygenin (21a) R1 = R2 = H, (Ð)-Matairesinol (91) R = Glc, (+)-Phillyrin (22a) R1 = Me, R2 = H, (Ð)-Arctigenin (92) R1 = Me, R2 = Glc, (Ð)-Arctiin

OMe OMe OH OH OH MeO OH O O OH HO Dirigent protein NADPH NADPH

Oxidase O HO OMe OMe OH HO HO OH OMe OMe (38) Coniferyl alcohol (3a) (+)-Pinoresinol (19a) (+)-Lariciresinol (20a) (Ð)-Secoisolariciresinol

NAD(P)

MeO MeO MeO O O O MeO MeO HO UDPGlc SAM O O O

OMe OMe OMe OGlc OH OH (92) (Ð)-Arctiin (22a) (Ð)-Arctigenin (21a) (Ð)-Matairesinol

Scheme 2

0[14[6[2[0 "¦#!Pinoresinol:"¦#!lariciresinol reductase The _rst enzyme identi_ed in the lignan pathway was the bifunctional\ NADPH!dependent\ "¦#! pinoresinol:"¦#!lariciresinol reductase[77\009\000 This circa 24 kDa enzyme was puri_ed "×2999!fold# to apparent homogeneity from F[ intermedia\000 and shown to catalyze the enantiospeci_c conversion of "¦#!pinoresinol "2a# into "¦#!lariciresinol "08a#\ and "¦#!lariciresinol "08a# into "−#!seco! isolariciresinol "19a#^ the corresponding antipodes "2b# and "08b# did not serve as substrates[ It is a type A reductase\ as established using ð3R!2HŁ and ð3S!2HŁNADPH as cofactors\ since only the pro!R hydrogen on the nicotinamide ring of NADPH was abstracted and transferred to the lignan product[77 As shown in Scheme 2\ this hydride transfer from NADPH during furanofuran lignan reduction could occur in any one of three ways\ i[e[\ via direct hydride attack onto the furano ring or the regenerated quinone methide\ resulting in either retention or inversion of apparent con_guration at C!6:C!6?[ Alternatively\ hydride attack could occur at either face and result in racemization[ That an {{inversion|| of con_guration mechanism occurred was established by exam! 553 Li`nans] Biosynthesis and Function

1 ination of the products obtained following individual incubation of "2#!ð6\6?! H1Łpinoresinols "2a\b# 1 and ð6\6?! H2Łlariciresinols "08a\b# with "¦#!pinoresinol:"¦#!lariciresinol reductase\ in the presence of NADPH "0[5 mM#] 0H NMR spectroscopic analyses "Figure 01# of the enzymatically generated lariciresinol "08# and secoisolariciresinol "19# revealed that hydride "deuteride# transfer occurred in

Figure 01 Partial 0H NMR spectra of lariciresinol "08# showing spectral regions for C!6?\ C!7?\ and C!7 proton resonances ""a# and "b## and secoisolariciresinol "19# showing spectral regions for C!6 and C!8 proton resonances ""c# and "d##[ "a# Synthetic "2#!lariciresinols "08a\b#\ "b# enzymatically synthesized "¦#!ð6\6?S! 1 1 H1Łlariciresinol "08a# obtained following incubation with "2#!ð6\6?! H1Łpinoresinols "2a\b#\ "c# synthetic "2#! 1 secoisolariciresinols "19a\b#\ and "d# enzymatically synthesized "−#!ð6\6?! H2Łsecoisolariciresinol "19a# obtained 1 77 following incubation with "2#!ð6\6?S! H2Łlariciresinols "08a\b#[

HR Ar Enz O HS H H HO H H H Ar O HO O H MeO H "Inversion" H OMe HO H HR 7' O Ar O H OH O MeO H or H 7 HS H H OMe Enz H Ar H HO O OH H "Retention" (3a) (+)-Pinoresinol O H MeO HO H HR Ar H OMe O HS H H H Ar OH HO Non-stereospecific H = C-7/C-7' protons of (+)-pinoresinol (3a) and (+)-lariciresinol (19a) (19a) (+)-Lariciresinol Ar = 4-hydroxy-3-methoxyphenyl Scheme 3 Li`nans] Biosynthesis and Function 554 a completely stereospeci_c manner\ with the incoming hydride "deuteride# taking up the pro! 1 1 R position to give "¦#!ð6\6?S! H1Łlariciresinol "08a# and "−#!ð6\6?S! H2Łsecoisolariciresinol "19a#\ respectively\ i[e[\ with {{inversion|| of con_guration at C!6 and C!6?[ The nucleotide sequence for the gene encoding "¦#!pinoresinol:"¦#!lariciresinol reductase has a single open reading frame encoding a polypeptide of 201 amino acids "Figure 02#\ giving a calculated molecular mass of 23[8 kDa[000 The authenticity of its sequence was established via assay of func! tionally recombinant "¦#!pinoresinol:"¦#!lariciresinol reductase\ as either its b!galactosidase fusion protein000 or the {{native|| protein in a pSBETa001 vector system expressed in E[ coli] both recombinant proteins catalyzed the enantiospeci_c conversion of "¦#!pinoresinol "2a# into "¦#!lariciresinol "08a#\ and the latter into "−#!secoisolariciresinol "19a#] the corresponding antipodes were not used as substrates "Figure 03#[ Sequence analysis of the cDNA also revealed that the NADPH!binding domain was situated close to the N!terminus\ consisting of three conserved glycine "as GXGXXG\ with X representing any residue# and four conserved hydrophobic residues "underlined in Figure 02#^ these are required for correct packaging for domain formation[002 The sequence also revealed considerable homology "52[4) to 50[5) similarity and 33[3) to 30[2) identity# to iso~avone reductases "see Table 3#\ which catalyze the analogous reduction of a\b unsaturated ketones during iso~avonoid formation[000 For example\ Pisum sativum iso~avone reductase converts 1?!hydroxy! pseudobaptigenin "82# into "2R#!sophorol "83#\ a precursor of the phytoalexin\ "¦#!pisatin "84# "Scheme 3#[ Moreover\ given that pinoresinol!derived lignans appear to be found in even the most primitive extant plants "e[g[\ ferns\ gymnosperms#\ whereas the iso~avonoids may be more restricted "e[g[\ to the Leguminosae#\ it is tempting to speculate that pinoresinol:lariciresinol reductase is the evolutionary forerunner of the iso~avonoid reductases[23 "¦#!Pinoresinol:"¦#!lariciresinol reductase also contains _ve conserved possible phosphorylation sites\ including Thr!291 "casein kinase II!type protein phosphorylation site# suggesting that its activity might be regulated by protein kinase cascades^23\000 a somewhat analogous situation also holds for iso~avonoid reductase"s#[ In agreement with these _ndings\ "¦#!pinoresinol:"¦#!lariciresinol reductase has also been detected in cell!free extracts from Forsythia koreana by Umezawa et al[003

Table 3 Homology between "¦#!pinoresinol:"¦#!larici! resinol reductase from Forsythia intermedia and iso~avone reductases from three legumes[000 *ÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐ Plant species Similarity ")# Identity ")# *ÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐ Cicer arietinum 52[4 33[3 Medica`o sativa 51[5 31[9 Pisum sativum 50[5 30[2 *ÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐ

0[14[6[2[1 "−#!Secoisolariciresinol dehydrogenase This NAD"P#¦!requiring enzyme catalyzes the enantiospeci_c conversion of "−#!seco! isolariciresinol "19a# into "−#!matairesinol "10a#[004\005 It has been puri_ed "× 5999!fold# to apparent homogeneity from F[ intermedia stem tissue\ and is an ½21 kDa protein\ as estimated by SDS! PAGE analysis[006\007 Interestingly\ the presumed lactol "85# intermediate was not detected in any of

MeO O HO O HO O Isoflavone OH reductase H O O H NADPH O O O O HO O HO O O (93) 2-Hydroxypseudobaptigenin (94) (3R)-Sophorol (95) (+)-Pisatin

Scheme 4 555 Li`nans] Biosynthesis and Function

Figure 02 Complete sequence of F[ intermedia "¦#!pinoresinol:"¦#!lariciresinol reductase cDNA plr!Fi0[ The NADPH binding domain conserved residues are underlined[ The _ve possible phosphorylation sites that are conserved among "¦#!pinoresinol:"¦#!lariciresinol reductase and the characterized iso~avone reductases are double underlined[ The stop codon is indicated by an asterisk[000

the enzyme assays using native protein "Scheme 4#[ The genes encoding "−#!secoisolariciresinol dehydrogenase were obtained using a PCR!guided strategy\ coupled with the screening of the F[ intermedia cDNA library[ Two of the _ve cDNA clones obtained\ SMDEHY520 and DEHY029\ were subsequently expressed in E[ coli\ and both gave proteins capable of enantiospeci_cally converting "−#!secoisolariciresinol "19a# into "−#!matairesinol "10a#[ Both had single open!reading frames encoding polypeptides of 161 amino acids\ giving calculated molecular masses of 18 kDa\ and isoelectric points of ½5[0[ However\ in contrast to the native dehydrogenase\ the corresponding lactol "85# in each instance was observed as an intermediate\ during conversion of "−#!seco! isolariciresinol "19a# into "−#!matairesinol "10a# "Scheme 4#[ Li`nans] Biosynthesis and Function 556

Figure 03 Chiral column HPLC analysis of lignans "a# lariciresinol "08# and "b# secoisolariciresinol "19#[ "2#!Pinoresinols "2a\b# were incubated with recombinant "¦#!pinoresinol:"¦#!lariciresinol reductase in the presence of ð3R!2HŁNADPH\ with unlabeled "2#!lariciresinols "08a\b# and "2#!secoisolariciresinols "19a\b# added as radiochemical carriers[ The solid line represents the UV absorbance trace "179 nm#\ whereas the dashed line shows that radioactivity is only incorporated into "¦#!lariciresinol "08a# and "−#!secoisolariciresinol "19a#\ respectively[000 For elution conditions see Figure 2[

MeO MeO MeO OH O O OH HO HO HO OH O

OMe OMe OMe OH OH OH

(20a) (Ð)-Secoisolariciresinol (96) (Ð)-Lactol (21a) (Ð)-Matairesinol

Scheme 5

0[14[6[2[2 Matairesinol O!methyltransferase Cell!free extracts of F[ intermedia were examined in order to attempt to establish whether O! methylation of matairesinol "10# giving arctigenin "11# occurred in an enantio! and regiospeci_c manner[098 Incubation with "2#!ðAr!2HŁmatairesinols "10a\b#\ in the presence of S!adenosyl!L! methionine\ gave both radiolabeled arctigenin "11# and isoarctigenin "86# "Scheme 5#\ with the "−#!enantiomeric form being preferentially obtained in both cases "1]0 and 1[4]0\ respectively#\ i[e[\ O!methylation was neither regio! nor stereospeci_c[ As for eudesmin "72# biosynthesis\ puri_cation of the actual O!methyltransferase involved in catalyzing the formation of "−#!arctigenin "11a#is required in order to determine whether the O!methyltransferase is truly enantio! and regiospeci_c[

MeO MeO MeO O O O MeO HO HO O O O SAM SAM

OMe OMe OMe OH OH OMe (22) Arctigenin (21) Matairesinol (97) Isoarctigenin

Scheme 6 557 Li`nans] Biosynthesis and Function 0[14[6[3 Linum usitatissimum] "−#!Pinoresinol:"−#!Lariciresinol Reductase and "¦#!Secoisolariciresinol Glucosyltransferase"s# Flaxseed "L[ usitatissimum\ Linaceae# has been used for several millennia for medicinal purposes[ There is considerable renewed interest in it because of the high levels "2Ð2[4)# of secoisolariciresinol diglucoside "87#\ which has an important role in the diet due to its protection against onset of breast and prostate cancers008\019 "see Section 0[14[03#\ and can be solubilized from ~axseed under very basic conditions "e[g[\ 9[2 M sodium methoxide#[010 Hydrolysis of secoisolariciresinol diglucoside "87# gives the aglycone "19#\ the chiral analysis "Chiralcel OD\ Daicel# of which revealed that essentially only the "¦#!antipode "19b# was present[86 The proposed biochemical pathway to secoisolariciresinol diglucoside "87# is shown in Scheme 6[ Preliminary experiments86 have indicated that stereoselective coupling of E!coniferyl alcohol "27# occurs to give "−#!pinoresinol "2b#[ Based on results from enzyme assays\ using cell!free extracts of developing seeds\ this is then reduced to _rst give "−#!lariciresinol "08b# and then "¦#!seco! isolariciresinol "19b#[ Screening procedures are under way to obtain the corresponding gene"s# for both the dirigent protein and reductase"s# from this source[ OMe OMe OH HO HO

O O Dirigent protein NADPH NADPH Oxidase O OH OMe OH OH OH OMe OMe

(38) Coniferyl alcohol (3b) (Ð)-Pinoresinol (19b) (Ð)-Lariciresinol

OMe OMe HO GlcO HO GlcO OH OH UDPGlc

MeO MeO OH OH (20b) (+)-Secoisolariciresinol (98) (+)-Secoisolariciresinol diglucoside

Scheme 7

Preliminary assays\ with partially puri_ed secoisolariciresinol glucosyltransferase\ in the presence of UDPð0!2HŁglucose and "2#!secoisolariciresinols "19a\b#\ have revealed that maximum activity of the glucosyltransferase occurs during the second week of seed development[

0[14[6[4 Thuja plicata and Tsuga heterophylla] Pinoresinol:Lariciresinol Reductases and Other Enzymatic Conversions Western red cedar "Thuja plicata# and western hemlock "Tsu`a heterophylla# are two long!living gymnosperms\ with the former having life spans in excess of 2999 years[ Of the two species\ cedar heartwood is particularly valued for its color\ durability\ and texture\ this in part being due to the massive deposition "½19) of dry weight# of heartwood "oligomeric# lignans\ derived from plicatic acid "64# and its congeners[24 In a somewhat analogous manner\ western hemlock accumulates various lignans\ such as 6?!hydroxymatairesinol "88# "9[2) in sapwood# and a!conidendrin "65# "9[94) in sapwood and 9[04 to 9[1) in heartwood#[011 Based on their respective chemical structures\ and the results from studies in the authors| laboratory\ it was rationalized that both plicatic acid "64# and a!conidendrin "65# were pinoresinol!derived "Scheme 7#[ Plicatic acid "64# could result from Li`nans] Biosynthesis and Function 558 matairesinol "10# via ring closure "aryltetrahydronaphthalene ring formation#\ hydroxylations "at both aromatic and aliphatic positions#\ and lactone ring cleavage\ whereas a!conidendrin "65# might result from regiospeci_c hydroxylation at the 6?!position of matairesinol "10# to give 6?! hydroxymatairesinol "88#\ with subsequent ring closure[ It was important to establish\ therefore\ whether the corresponding pinoresinol:lariciresinol reductases were present in these plant systems in addition to the dirigent proteins "see Section 0[14[5[3#[

O O 67 MeO 51 8 9 MeO 8 O O 7' 7' 4 2 HO 8' 9' HO 8' 3 HO 1' 6' 2' 3' 5' 4' OMe OMe OH OH (76) α-conidendrin (99) 7'-Hydroxymatairesinol OMe OH

O O MeO O O HO

OMe OMe (3) Pinoresinol OH (21) Matairesinol

OMe

OH MeO MeO OH O O OH HO HO O

HO OMe OMe HO OH OH OMe (20) Secoisolariciresinol (21) Matairesinol (19) Lariciresinol Ring closure (aryltetrahydronaphthalene lignan formation) Hydroxylation steps Lactone cleavage

OH MeO OH

HO CO2H OH

MeO OH OH (75) Plicatic acid

Scheme 8 569 Li`nans] Biosynthesis and Function Thus\ screening of a western red cedar cDNA library\ via a PCR!guided strategy\ gave two pinoresinol:lariciresinol reductase cDNAs with ½47) identity and ½69) similarity to plr!Fi0[ Their recombinant proteins\ when expressed in E[ coli\ displayed distinct pinoresinol:lariciresinol reductase activities[29\012\013 The _rst\ plr!Tp0\ catalyzed the NADPH!dependent conversion of "−#! pinoresinol "2b# into "−#!lariciresinol "08b#\ this being subsequently reduced to "¦#!seco! isolariciresinol "19b# "see Scheme 8#\ with the enantiospeci_city of the conversion being determined by chiral HPLC analysis of the enzymatic products so obtained "Figure 04"a##[ The second\ plr! Tp1\ isolated from the same plant source\ converted "¦#!pinoresinol "2a# into "¦#!lariciresinol "08a#\ and then into "−#!secoisolariciresinol "19a# "see Scheme 09 and Figure 04"b##[ That is\ western red cedar has genes encoding two reductive pathways with di}ering enantiospeci_cities[ Although the biological signi_cance of both pathways needs to be established\ this study reveals that the pathway to plicatic acid "64# indeed occurs via involvement of both dirigent proteins and pinoresinol: lariciresinol reductase"s#[

OMe OMe

HO HO OH OMe O HO NADPH NADPH

plr-Tp1 plr-Tp1 HO O O OH OH OH OH MeO OMe OMe (3b) (Ð)-Pinoresinol (19b) (Ð)-Lariciresinol (20b) (+)-Secoisolariciresinol

Scheme 9

Figure 04 Chiral column HPLC analysis of the secoisolariciresinol "19# obtained after incubation of "2#!ð2\2?! 03 O CH2Łpinoresinols "2a\b# with recombinant pinoresinol:lariciresinol reductase\ in the presence of NADPH[ "a# plr!Tp0\ and "b# plr!Tp1[ Unlabeled "2#!secoisolariciresinols "19a\b# were added as radiochemical carriers[ The solid line represents the absorbance trace at 179nm\ whereas the dashed line shows the radioactivity incorporated into the corresponding secoisol ariciresinol "19# product[

Further characterization of the recombinant reductases\ plr!Tp0 and plr!Tp1\ resulted in the discovery that both di}er substantially from their Forsythia counterpart] plr!Tp0 preferentially converts "−#!pinoresinol "2b# into "−#!lariciresinol "08b#\ but can also slowly reduce "¦#!pinoresinol "2a# to give "¦#!lariciresinol "08a# although the latter is not further metabolized[ The converse situation was observed for plr!Tp1[ It preferentially converts "¦#!pinoresinol "2a# into "¦#!lari! ciresinol "08a#\ but can also slowly reduce "−#!pinoresinol "2b# into "−#!lariciresinol "08b#[ As for plr!Tp0\ plr!Tp1 is fully enantiospeci_c for lariciresinol "08# reduction\ since it only utilizes "08a#as a substrate and not its "−#!enantiomer "08b#[29\013 In a comparable manner\ a T[ heterophylla cDNA library was screened\ this resulting in two Li`nans] Biosynthesis and Function 560 OMe OMe OH OH MeO OH O O OH HO NADPH NADPH

plr-Tp2 plr-Tp2 O HO OMe

HO HO OH OMe OMe (3a) (+)-Pinoresinol (19a) (+)-Lariciresinol (20a) (Ð)-Secoisolariciresinol

Scheme 10

putative cDNAs encoding pinoresinol:lariciresinol reductase with ½62) similarity and ½51) identity[ Taken together\ these _ndings may help explain the results of Umezawa and Shimada where the formation of secoisolariciresinol "19#\ catalyzed by cell!free extracts from Arctium lappa petioles\ was investigated[014 In that study\ coniferyl alcohol "27# was incubated with the cell!free preparation in the presence of H1O1 and NADPH[ Under these conditions\ nonspeci_c peroxidase catalyzed coupling occurs to give the racemic lignans\ "2#!"4a\b#\ "2#!"5a\b#\ and "2#!"2a\b#\ with the latter then undergoing NAPDH!dependent reduction to a}ord secoisolariciresinol "19#\ whose "¦#!enanti! omer "19b# was in enantiomeric excess "e[e[ 19)#[ Additionally\ "¦#!secoisolariciresinol "19b# was isolated from MeOH extracts of A[ lappa petioles in 67) enantiomeric excess[014 These data are consistent with both "¦#! and "−#!pinoresinol:lariciresinol reductases being present in this species\ as previously described for western red cedar[

0[14[6[5 Linum ~avum and Podophyllum hexandrum] Podophyllotoxin and its Pinoresinol Precursor "−#!Podophyllotoxin "12# accumulates up to 3) of the dry weight in P[ hexandrum "Podo! phyllaceae# rhizomes015 and "−#!4!methoxypodophyllotoxin "099# up to 2[4) of the dry weight in L[ ~avum "Linaceae# roots[016 Podophyllotoxin "12#\ as its etoposide "090# and teniposide "091# derivatives\ is used in treatment of various cancers "see Section 0[14[03[1#[ Because of the limited supply of Podophyllum rhizomes\ due to their intensive collection in the wild\ there is a growing interest in de_ning and exploiting the podophyllotoxin "12# biosynthetic pathway[ This is in part because studies\ using both cell suspension and root tissue cultures\ have not been very successful in producing signi_cantly elevated levels of either podophyllotoxin "12# or "−#!4!methoxy! podophyllotoxin "099#[016Ð021 Accordingly\ both de_ning the pathway and establishing what factors a}ect its induction seem to be worthwhile goals in preparation for future biotechnological manipu! lations[

H H

R OH O O O O S O O O 8 HO HO HO O HO O O O 8 O 8 O 8' O O O O 8' O 8' O O MeO OMe OMe MeO OMe MeO OMe OH OH (23) R = H, (Ð)-Podophyllotoxin (101) Etoposide (102) Teniposide (100) R = OMe, (Ð)-5-Methoxypodophyllotoxin 561 Li`nans] Biosynthesis and Function Preliminary investigations by Broomhead et al[ suggested that "−#!matairesinol "10a# might be a precursor of podophyllotoxin "12#[022 Accordingly\ it was important to establish whether the bio! chemical pathway to matairesinol "10# was also present in L[ ~avum[ Thus\ cell!free extracts from L[ ~avum roots were incubated with "2#!pinoresinols "2a\b# "19 mM# in the presence of NADPH "19 mM#\ where it was established that both "¦#!lariciresinol "08a# and "−#!secoisolariciresinol "19a# were formed[023 Furthermore\ a partially puri_ed secoisolariciresinol dehydrogenase preparation was also obtained which\ when incubated with "2#!ðAr!1HŁsecoisolariciresinols "19a\b# and NAD\ only gave "−#!ðAr!1HŁmatairesinol "10a#[ The enzymatically formed "−#!ðAr!1HŁmatairesinol "10a# had a cluster of ions centered at m:z 259 "Figure 05"a## relative to that of "2#!ðAr!1HŁsecoisolariciresinol "19a\b# cluster of ions at m:z 253 "Figure 05"b##\ thereby establishing the intact enzymatic conversion\ and thus veri_cation of the pathway to "−#!matairesinol "10a# in this species[

Figure 05 Mass spectra of "a# "−#!ðAr!1HŁmatairesinol "10a# obtained following incubation with a partially puri_ed secoisolariciresinol dehydrogenase preparation of Linum ~avum and "2#!ðAr!1HŁsecoisolariciresinols "19a\b# in the presence of NAD\ "b# "2#!ðAr!1HŁsecoisolariciresinol "19a\b# substrates[

Thus\ in summary\ pinoresinol "2# formation and metabolism have been shown to occur in Forsythia species\ Linum usitatissimum\ Thuja plicata\ Tsu`a heterophylla\ Linum ~avum\ and Podo! phyllum peltatum\ thereby establishing its pivotal role in 707? linked lignan biosynthesis[

0[14[7 ARE DIRIGENT PROTEIN HOMOLOGUES INVOLVED IN OTHER 707? PHENOXY RADICAL COUPLING PROCESSES< In addition to coniferyl alcohol "27# derived 707? linked lignans\ there are optically active 707? linked lignans which provisionally appear to result from coupling of other monomeric precursors\ such as E!p!coumaryl alcohol "21#\ E!sinapyl alcohol "33#\ various hydroxycinnamic acids: allylphenols\ and their derivatives[ Although each of their biochemical pathways await delineation\ the occurrence of these lignans suggests the presence of dirigent proteins with di}ering substrate speci_cities[ This\ in turn\ is consistent with the existence of an entire new class of proteins\ and this possibility is discussed below using just a few examples for illustrative purposes only[

0[14[7[0 Ligballinol "p!Coumarylresinol# and Related Structures Based on the chemical structures of ligballinol " p!coumarylresinol# "092#\ termilignan "093#\ anolignan B "094#\ and thannilignan "095#\ it can be proposed that all are either p!coumaryl alcohol "21#orp!hydroxyarylpropene "59# derived[ Of these\ ligballinol "p!coumarylresinol# "092# was _rst isolated in optically active form from squirting cucumber "Ecballium elaterium\ Cucurbitaceae# fruits\024 but also accumulates in enantiomeric excess in red bean "Vi`na an`ularis\ Leguminosae# cell suspension cultures treated with actinomycin D[025 Interestingly\ although both isolates have very distinct ðaŁD values suggesting di}erent enantiomeric compositions\ the chiral HPLC analyses of the ligballinol "p!coumarylresinol# "092# preparations have not been reported[ Termilignan Li`nans] Biosynthesis and Function 562 "093#\ anolignan B "094#\ and the optically active thannilignan "095#\ from Terminalia bellerica "Combretaceae#\026 can also be proposed to partly result from regiospeci_c:stereoselective E!p! coumaryl alcohol "21# coupling\ although they might also derive from the corresponding allylphenol "59#[ Whether such coupling is under the control of dirigent!like proteins awaits delineation\ as does identi_cation of the nature of the precursors involved[

O (103) Ligballinol = p-coumarylresinol 8' (Ecballium elaterium), [α] = Ð7.1¡ (MeOH, c = 1.61) 8 D 28 (Vigna angularis), [α]D = +25.3¡ (MeOH, c = 0.06) O

HO OH OMe 1 9' CH2 R HO 8' 8' 8 8 CH2 CH2 OH R2 HO (104) R1 = OH, R2 = OMe, Termilignan (106) (Ð)-Thannilignan 1 2 24 (105) R = H, R = OH, Anolignan B [α]D = Ð63.9¡ (CHCl3, c = 0.3) (Terminalia bellerica) (Terminalia bellerica)

0[14[7[1 Syringaresinol and Medioresinol As summarized in Table 0\ syringaresinol "55# is found in di}erent species\ with predictably di}erent degrees of enantiomeric purity depending upon the plant species involved[ Its formation could result from either dirigent protein mediated coupling of sinapyl alcohol "33#\ albeit with more than one mode of stereoselective coupling to a}ord both "¦#! and "−#!enantiomers "55a# and "55b#\ respectively\ or perhaps less likely\ it could result from modi_cation of preformed pinoresinol "2#[ As an added consideration\ optically active medioresinol "096# present in Eucommia ulmoides "Eucommiaceae#\027 might result from either heterogeneous coupling of E!coniferyl "27# and sinapyl "33# alcohols\ or be formed through pinoresinol "2# modi_cation "Scheme 00#[ Accordingly\ the precise enzymology involved in their formation awaits full clari_cation\ including that of the role of dirigent proteins[

0[14[7[2 Pellia Liverwort Lignans Lignans present in liverworts\42Ð59 such as Pellia epiphylla\ appear to be formed via stereoselective coupling of E!ca}eic acid "22# molecules\ as shown in Scheme 01[ Thus\ following stereoselective coupling\ the regenerated diphenol on ring A can then participate in nucleophilic attack onto the quinone methide of ring B to generate the resulting aryldihydronaphthalene derivative "34#[ Although the enantiomeric composition of these lignans has yet to be described\ their pronounced optical rotations suggest involvement of dirigent proteins[

0[14[7[3 Lignanamides Various plant species\ particularly in\ but not restricted to\ the Solanaceae accumulate p!coum! aroyl and feruloyl tyramine derivatives "097# and "098#\ with these being considered to be incor! porated "at least in part# into the aromatic component of the suberin biopolymer[028Ð030 Feruloyl "098#\ sinapoyl "009#\ and ca}eoyl "000# tyramine derivatives\ and the optically active aryl! dihydronaphthalene lignanamides "001# and "002#\ have also been isolated from Porcelia macrocarpa 563 Li`nans] Biosynthesis and Function OMe OH OH

O OMe Dirigent protein

Oxidase MeO O MeO OMe OH HO OMe (44) Sinapyl alcohol (66) Syringaresinol

OMe OH OH OH

O OMe Dirigent protein + Oxidase O MeO OMe OMe OH OH HO (44) Sinapyl alcohol (38) Coniferyl alcohol OMe (107) Medioresinol

? OMe OMe OH OH OH

O O OMe Dirigent protein ?

Oxidase MeO O O OMe OH HO HO (38) Coniferyl alcohol OMe OMe (3) Pinoresinol (66) Syringaresinol

Scheme 11

"Annonaceae# branch tissue[031 It is\ accordingly\ again tempting to speculate that the lignanamides result from dirigent protein mediated stereoselective coupling "cf[ the liverwort lignans#[ Fruits of Xylopia aethiopica "Annonaceae# also contain optically active 707? linked "−#!can! nabisin B "003# and "−#!cannabisin D "004#\ together with smaller amounts of the racemic lignans\ "2#!grossamide "005# and "2#!demethylgrossamide "006#[032 Perhaps signi_cantly\ the small amounts of racemic products "005# and "006# co!occur with larger amounts of the corresponding {{monomeric|| tyramine derivatives "097#\ "098#\ and "000#[ Indeed\ based on the methodologies employed for their isolation "plant tissue grinding over long periods\ lengthy extractions\ numerous chromatographic steps#\ formation of the racemic dimers could result from nonspeci_c coupling "artifact formation# during isolation[ This is because during their isolation disruption of the plant material will result in the tyramine derivatives "097#\ "098#\ and "000# coming into contact with nonspeci_c oxidases:oxidants[ Surprisingly\ few studies seem to consider the possibility that isolated "racemic# phenolic dimers "and higher oligomers# may be artifacts\ in spite of the readily oxidizable nature of such compounds[ Li`nans] Biosynthesis and Function 564

H HO CO2H HO CO2H HO CO2H ¥ A One electron HO oxidation O O CO2H

HO CO2H Dirigent HO ¥ CO2H protein B

HO O OH O (33) Caffeic acid

+ : HO CO H 2 HO CO2H HO CO2H A A A HO CO H 2 HO CO2H HO CO2H H

B B B OH OH OH OH OH O (45) 20 [α]D = Ð130.77¡ (MeOH) (Pellia epiphylla) Scheme 12

OH O (108) R1 = R2 = H, p-Coumaroyl tyramine R1 N (109) R1 = OMe, R2 = H, Feruloyl tyramine H (110) R1 = R2 = OMe, Sinapoyl tyramine HO (111) R1 = OH, R2 = H, Caffeoyl tyramine R2

OH OH O O H H MeO 8 MeO 8 N N N N HO 8' HO 8' H H OMe O O OH OH

MeO OMe OH OH OH (112) (113) 25 25 [α]D = Ð20¡ (MeOH, c = 0.062) [α]D = Ð12¡ (MeOH, c = 0.085) (Porcelia macrocarpa) (Porcelia macrocarpa)

OH HO O O H OMe RO 8 N N H N HO 8' H 5' O H O N OH 8 O OR OR HO

OH OH

(114) R = H, Cannabisin B, [α]D = Ð38¡ (MeOH, c = 0.25) (116) R = Me, (±)-Grossamide (115) R = Me, Cannabisin D, [α]D = Ð46¡ (MeOH, c = 0.25) (117) R = H, (±)-Demethylgrossamide 565 Li`nans] Biosynthesis and Function Cannabis sativa "Cannabidaceae# fruits033Ð035 also reputedly contain racemic 707? ""003#\ "004#\ "007##\ 70O03? ""008#\ "019##\ and 704? ""005#\ "006## linked lignan amides\ together with their {{monomeric|| precursors "097#\ "098#\ and "000#[ At a _rst glance\ the occurrence of these dimers would appear to favor random coupling mechanisms in vivo[ However\ such reports of racemic products being present in plant tissues must be viewed with some caution\ since once again the isolation conditions employed were very harsh\ and it cannot be ruled out that these {{randomly linked|| dimers are instead just isolation artifacts[

OH O OH H OH O HO 8 N MeO 8 N N H HO 8' O 4' H HO O H OH N MeO OH O OH OH (118) Cannabisin A (119) Cannabisin E

OH O

MeO 8 N H O 4' HO H N MeO O OH (120) Cannabisin F

Artifact formation may also account for the occurrence of racemic grossamide "005# in bell pepper "Capsicum annuum var[ `rossum\ Solanaceae# roots[036 In this case\ homogenized roots "10 kg# were extracted for 225 h with 69) EtOH "059 L#\ prior to any chromatographic puri_cation\ i[e[\ under conditions which would certainly favor nonspeci_c coupling and artifact formation from the monomeric precursors present in the tissue[

0[14[7[4 Guaiaretic Acid\ Steganacin\ and Gomisin A Certain plant species contain relatively unusual types of optically active 707?\ as well as 707?\ 101? linked lignans that appear to be derived via either allylphenol and:or monolignol coupling[ Examples include the 707? linked "−#!guaiaretic acid "0# from Guaiacum of_cinale\09 "−#!steganacin "14# from Ste`anotaenia araliacea "Umbelliferae#\037 and "¦#!gomisin A "010# from the fruits of Schizandra chinensis "Schizandraceae#[038\049 The formation of these optically active lignans is again of considerable interest] "−#!guaiaretic acid "0# can be envisaged to result via stereoselective coupling of "iso#eugenol "47:48# "cf[ the mechanism shown in Scheme 01#\ in a process presumably mediated by the corresponding dirigent protein[ "−#!Steganacin "14#\ on the other hand\ could result from dirigent protein mediated coupling to give "¦#!pinoresinol "2a#\ this being metabolized into "−#!matairesinol "10a# or a substituted butyrolactone equivalent "Scheme 02#[ A most interesting biochemical conversion then follows which stipulates 101? intramolecular coupling] this may involve a cytochrome P349 catalyzed process\ in a manner comparable to diphenol intramolecular coupling processes leading to the alkaloids "R\S#!berbamunine\ "R\S#!norberbamunine\ and "R\R#!guattegaumerine from "S#!coclau! rine\ "R#!N!methylcoclaurine\ and "S#!N!methylcoclaurine\ respectively\ in Berberis stolonifera plant cell cultures[040 "¦#!Gomisin A "010# appears to be derived from dirigent protein mediated stereoselective coup! ling of "iso#eugenol "47:48# "Scheme 03#[ If correct\ this would initially a}ord the bis!quinone methide which could then be acted upon by the corresponding reductase to generate the putative Li`nans] Biosynthesis and Function 566

2' MeO 8' O AcO O 8' 8 O MeO O O O HO 8 8 8' 2' 2 O HO O 2 2 2' MeO MeO OMe OH HO MeO OMe (21) Matairesinol or a 2Ð2' intramolecular coupling (25) (Ð)-Steganacin substituted butyrolactone equivalent [α]D = Ð122.6¡ (CHCl3, c 1.02) (Steganotaenia araliacea)

Scheme 13 dibenzylbutane "011#[ The latter "or some equivalent# could then undergo 101? coupling\ as well as various hydroxylation\ methylation\ and methylenedioxy bridge formation steps\ to ultimately a}ord "¦#!gomisin A "010#[

MeO MeO ¥ ¥ oxidative O O coupling

dirigent protein OMe O O OH OMe OMe (59) Isoeugenol

O MeO O

HO 8 MeO 2 8' MeO 2' OH OMe MeO

OH MeO (122) (121) (+)-Gomisin A [α]D = +67.9¡ (CHCl3) (Schizandra chinensis) Scheme 14

0[14[8 MISCELLANEOUS COUPLING MODES] ARE DIRIGENT PROTEINS ALSO INVOLVED< Consideration of the chemical structures and optical rotations of many other isolated lignans strongly suggests involvement of dirigent proteins stipulating distinct coupling modes[ This is illustrated with only three examples\ namely the 701? linked "−#!blechnic acid "52# from the fern B[ orientale\53 the 704? linked "¦#!denudatin B "012# from Ma`nolia denudata "Magnoliaceae#041 and the 700? linked "−#!megaphone "3# from Aniba me`aphylla "Lauraceae#[042

MeO OMe 8 MeO 5' O O (123) (+)-Denudatin B [α]D = +82.7¡ (MeOH, c = 2.67) (Magnolia denudata) 567 Li`nans] Biosynthesis and Function Blechnic acid "52#\ which reputedly co!occurs with its epimeric form\ 6!epiblechnic acid "013#\ and other conjugates in B[ orientale\ provisionally appears to result from stereoselective coupling of two ca}eic acid "22# molecules[ Its formation can be envisaged to occur through the involvement of a dirigent protein!mediated process in a manner analogous to that for "¦#!pinoresinol "2a# formation in Forsythia species\ with the proposed biosynthetic scheme shown in Scheme 04[ In a somewhat analogous manner\ formation of both "¦#!denudatin B "012# and "−#!megaphone "3# can be envisaged to occur via stereoselective 704? and 700? coupling of two "iso#eugenol "47:48# derived compounds\ followed by skeletal modi_cations as needed[ Accordingly\ these and many other examples again strongly suggest that a class of dirigent proteins exists with each engendering distinct coupling modes[

OH O OH

2' 8 CO2H OH

CO2H (124) (Ð)-7-Epiblechnic acid 23 [α]D = Ð145¡ (MeOH, c = 1.0) (Blechnum orientale)

CO2H CO H CO2H 2 ¥ O CO H oxidative HO H ¥ 2 coupling O OH dirigent protein OH HO CO H O 2 OH O H OH (33) Caffeic acid

O OH OH OH : OH O OH OH 2' 8 CO2H CO2H

CO2H CO2H (63) (Ð)-Blechnic acid or (64) epimer Scheme 15

0[14[09 704? AND 70O03? COUPLING OF MONOLIGNOLS AND ALLYLPHENOLS AND THEIR ASSOCIATED METABOLIC PROCESSES A number of plant species in the pteridophytes\ gymnosperms\ and angiosperms contain 704? and 70O03? linked lignans\ in addition to their 707? constituents[ These lignans can\ depending upon the plant species from which they are isolated\ exist in either optically pure or near racemic form[ Of these\ the most frequently reported are the dehydrodiconiferyl alcohols "4a# and "4b#\ the guaiacylglycerol 70O03? coniferyl alcohol ethers "5#\ and derivatives thereof[ Another example of an 704? linked lignan is that of the presumed allylphenol!derived lignan\ licarin A "46#[48\043 The Li`nans] Biosynthesis and Function 568 proposed main metabolic role of lignans "4# and "5# in vivo are as intermediates in lignin formation60 "but see Chapter 2[07#\ whereas other derivatives\ such as dehydrodiconiferyl alcohol glucosides "014a\b#\ are reputed to be cell wall components functioning as cytokinins044Ð046 "but see Section 0[14[02[4#[

8 OH 5' OH O O HO O MeO HO OH OMe (125a,b)

Prior to discussing the biochemical processes known to involve the common 704?:70O03? coupled products\ a brief discussion of their relative con_gurations and optical activities is required[ In a study by Hirai et al[\047 each enantiomer of dehydrodiconiferyl alcohol "4a\b# was separated and subsequently degraded by chemical means to give methylsuccinic acid "015# via aromatic ring _ssion "Scheme 05#[ Following analysis of the con_gurations of the resulting methylsuccinic acids "015a# and "015b# at carbon 1 "corresponding to C!7 of lignan "4##\ it was deduced that the "¦#! and "−#!forms of dehydrodiconiferyl alcohol "4a# and "4b# had 6S\7Rand 6R\7Scon_gurations\ 14 respectively[ Interestingly\ the enantiomers of dehydrodiconiferyl alcohol "4a# and "4b# have ðaŁD values of ¦25[2> "MeOH\ c 9[057# and −33[5> "MeOH\ c 9[075#\048 whereas the synthetic 14 6?07? dihydro derivatives "016a# and "016b# "Scheme 06# have lower ðaŁD values\ i[e[\ ¦4[0> "MeOH\ c 9[085# and −8[07> "MeOH\ c 9[133#\ respectively[ On the other hand\ phenols "017a# and 14 "017b#\ which have only a single chiral center due to 60O03? ether reductive cleavage\ have ðaŁD values of ¦14[73> "MeOH\ c 9[198# and −18[24> "MeOH\ c 9[073#[

CO2H 8 5' OH H 2 HO 7 O 4' HO2C MeO OMe (5a) (7S, 8R)-(+)-Dehydrodiconiferyl alcohol (126a) (R)-(+)-Methylsuccinic acid

CO2H 8 5' OH H 2 HO 7 O 4' HO2C MeO OMe (5b) (7R, 8S)-(Ð)-Dehydrodiconiferyl alcohol (126b) (S)-(Ð)-Methylsuccinic acid

Scheme 16

While most 0H and 02C NMR spectroscopic studies of 704? linked lignans have established a trans con_guration for the constituents attached to carbons 6 and 7\059Ð051 there are a number of studies which concluded that various 704? linked lignans were in a cis con_guration[55\052Ð054 Detailed analyses by Wallis and co!workers\ however\ established that only the trans isomers were natural products\ and that the proposed cis con_gurations resulted from misinterpretation of spectroscopic data[56 With this background regarding optical activities and con_gurations\ naturally occurring 704? linked lignans seem to be present in di}erent plants with di}ering degrees of enantiomeric purity\ based on their observed optical rotations\ e[g[\ "−#!licarin A "46# from the liverwort Jackiella 48 javanica\ which has an ðaŁD −32> "c 8[9#\ is presumed to be optically pure\ whereas the same compound "46#inEupomatia laurina is apparently racemic[055 Additionally\ the aglycone "016#of 10 trans!dihydrodehydrodiconiferyl alcohol glucoside "60#\ ðaŁD −7[4> "c 9[85\ acetone#\ from the fern\ P[ vittata\55 is also apparently in signi_cant enantiomeric excess\ based on comparison with the optical rotation data obtained for the synthetic analogue "016b# described above[ Optical 579 Li`nans] Biosynthesis and Function OH OH

8 8 5' OH Pd/C, EtOAc, H2 (1 atm) 5' OH HO HO 7 10 min 7 O 4' O 4' MeO OMe MeO OMe (5a,b) (±)-Dehydrodiconiferyl alcohols (127a,b) (±)-Dihydrodehydrodiconiferyl alcohols

Pd/C, EtOAc, H2 (1 atm) 10 min

HO OH

5' MeO 8 OH 7

HO 4' OMe (128a,b) Scheme 17

activities for numerous other 704? linked lignans have also been described in the gymnosperms and angiosperms[ In general\ however\ these have a wide range of optical rotations[ Finally\ the 70O03? linked lignans "5#\ which exist in vivo in both erythro and threo form\ have diastereomers that are readily separable by conventional chromatographic methodologies[76 Although the chiral separation of each form has not been reported\ enantiomeric separation was achieved with synthetic analogues\ i[e[\ erythro and threo forms of lignan "018#[05

HO 8 O 4' OMe

OMe OH (129) (±)-threo/erythro

0[14[09[0 Formation and Metabolism of 704? and 70O03? Linked Lignans As for the 707? linked lignans\ the fact that many 704? and 70O03? lignans are either enantiomerically pure or in enantiomeric excess requires an explicit biochemical explanation\ in terms of whether the coupling steps are stereoselective\ and:or if subsequent metabolic conversions are enantiospeci_c[ However\ since no systematic study has been reported\ it is premature to discuss in any detail how 70O03? and 704? coupling may be either regio! or stereoselectively controlled\ and whether dirigent protein mediation is involved[ On the other hand\ the optical activities noted for both "−#! licarin A "46# from J[ javanica48 and the "−#!aglycone "016b# from deglycosylation of "−#!trans! dihydrodehydrodiconiferyl alcohol glucoside "60# found in P[ vittata55 suggest that stereoselective coupling of "iso#eugenol "47:48# and coniferyl alcohol "27#\ respectively\ occurs*at least in these species*but this needs to be established at the protein:enzyme level[ Signi_cant progress has been made though in delineating postcoupling metabolic conversions of dehydrodiconiferyl alcohol "4# and guaiacylglycerol 70O03? coniferyl alcohol ether "5# in loblolly pine "Pinus taeda# cell suspension cultures[048\057Ð069 Not unexpectedly\ based on chemical charac! terization of previously isolated lignans in the Pinaceae\057Ð069 the most common modi_cations are the regiospeci_c reduction of the 6?07? allylic double bond of "4# and "5#\ and demethylation at carbon 2? "see compounds "016# and "029#Ð"021##[ Additional regiospeci_c transformations also Li`nans] Biosynthesis and Function 570 occur\ which involve 60O03? reduction "017#\ and in other species\ acylation of the 8 and 8? aliphatic hydroxyl groups with acetate\ p!coumaroyl\ and feruloyl moieties "022#Ð"025#[

OH OH O HO OH HO RO OH O MeO OR OMe OH (127a) R = Me, Dihydrodehydrodiconiferyl alcohol (131) R = H 25 (132) R = Me [α]D = +5.1¡ (MeOH, c = 0.6) 25 (Pinus massonian) a (130) R = H, Cedrusin, [α]D = +3.2¡ (MeOH, c = 1.0) (Pinus massonian) a

OAc AcO OAc

OAc MeO OAc AcO O AcO MeO OMe OMe (133) Tran s-Dihydrodehydrodiconiferyl alcohol triacetate (134) 25 25 [α]D = Ð66¡ (CHCl3, c = 0.8) [α]D = Ð2.5¡ (CHCl3 c = 2.0) (Cryptomeria japonic) a (Cryptomeria japonic) a

OR1

OR2 HO O MeO OMe

1 2 28 (135) R = R = p-coumaroyl, [α]D = +2.0¡ (MeOH, c = 0.5) 1 2 28 (136) R = p-coumaroyl, R = feruloyl, [α]D = +2.3¡ (MeOH, c = 0.5) (Corylus sieboldian) a

0[14[09[0[0 Phenylcoumaran 60O03? ring reduction The 704? linked lignans\ such as dehydrodiconiferyl alcohol "4# and licarin A "46#\ both contain phenylcoumaran ring structures\ which result from nucleophilic attack of the phenol group of ring A onto the quinone methide\ as shown in Scheme 07[ In certain gymnosperms\ such as Cryptomeria japonica\ there are other 704? linked lignans whose structures might be considered to result from reduction of the 60O03? benzylic ether of the phenylcoumaran ring[ In C[ japonica\ these 14 060 metabolites e[g[\ "023# have low optical rotations "ðaŁD −1[4>\ c 1[9\ CHCl2# suggesting that reductive cleavage might only be regio! rather than enantiospeci_c[ Such reductions are mech! anistically analogous to those for pinoresinol:lariciresinol and iso~avonoid reductases[ In this regard\ there have been a number of reports of so!called iso~avone reductase homologues\ which show considerable sequence homology to both iso~avone and pinoresinol:lariciresinol reductases\ e[g[\ from A[ thaliana\ Nicotiana tabacum\ Solanum tuberosum\ Zea mays\ and Lupinus albus "summarized by Dinkova!Kostova et al[000#[ None of these homologues have\ however\ any known catalytic function[ During screening of a P[ taeda cDNA library\ one such homologue was obtained\048 and Figure 06 shows its gene sequence[ The corresponding recombinant protein was subsequently expressed in E[ coli\ but was unable to reduce "2#!pinoresinols "2a\b#[ On the other hand\ when incubated with 571 Li`nans] Biosynthesis and Function R R R R A R : 2 HO HO OMe O O OMe MeO OMe OMe OH

(38) R = CH2OH, Coniferyl alcohol (5) R = CH2OH, Dehydrodiconiferyl alcohol (59) R = Me, Isoeugenol (57) R = Me, Licarin A Scheme 18

"2#!dehydrodiconiferyl alcohols "4a\b#\ it catalyzed 60O03? reduction of both enantiomers to give the products "017a\b#\061 albeit at low speci_c activity compared with pinoresinol:lariciresinol reductase[ As for pinoresinol:lariciresinol and iso~avone reductases\ it is a type A reductase\ since only the 3 pro!R\ but not the 3 pro!S\ hydride is abstracted from NADPH during reductive cleavage[ In contrast to the enantiospeci_c Forsythia pinoresinol:lariciresinol reductase\ the recombinant 60O03? reductase was capable of e}ectively reducing both "¦#! and "−#!antipodes "4a# and "4b#[

Figure 06 Complete sequence of Pinus taeda 60O03? !reductase cDNA\ plrh!Pt[

Thus\ it appears there is a family of reductases in phenylpropanoid metabolism\ which catalyze the reduction of iso~avonoids "e[g[\ hydroxypseudobaptigenin "82##\ 707? linked lignans "e[g[\ pinoresinol "2# and lariciresinol "08##\ and 704? linked phenylcoumarans "e[g[\ dehydrodiconiferyl alcohol "4##[ The latter\ however\ di}ers from the others by the ability to e}ectively reduce both "¦#! and "−#!antipodes "4a# and "4b# of the substrate\ i[e[\ it is regio! rather than enantiospeci_c[

0[14[09[0[1 Allylic 607? bond reduction A common structural modi_cation of 704? and 70O03? lignans in planta\ including in the Pinaceae such as Pinus taeda cell suspension cultures\ is that of the so!called {{dihydro|| derivatives\ whose structures are based mainly on dihydrodehydrodiconiferyl alcohol "016#059Ð051\053\054\057\060Ð074 and guaiacylglycerol 70O03? dihydroconiferyl alcohol ethers "021#[054\061\067Ð079 However\ in addition to these substances\ other {{dihydro|| compounds frequently co!occur\ including dihydro! p!coumaryl "026#067 and dihydroconiferyl "027#051\054\062\065\067 alcohols\ as well as various dihydro! cinnamic acids e[g[\ "028# and "039#[062\067 Li`nans] Biosynthesis and Function 572

R2 OH 1 2 (137) R = CH2OH, R = H, Dihydro p-coumaryl alcohol 1 2 (138) R = CH2OH, R = OMe, Dihydroconiferyl alcohol 1 2 (139) R = CO2H, R = H, Dihydro p-coumaric acid 1 2 (140) R = CO2H, R = OMe, Dihydroferulic acid

P[ taeda suspension cultures have been used to de_ne the biochemical processes involved in allylic double bond reductions of dehydrodiconiferyl alcohols "4a\b#\ where using partially puri_ed reductase preparations\ it was established that NADPH "but not NADH# was required as a cofactor[058 Individual incubation of "4a\b# with ð3R!2HŁ and ð3S!2HŁNADPH\ respectively\ revealed that only the 3 pro!R hydride was transferred to the reduced product "016#\ with the 6?07? allylic bond reductase being capable of utilizing both "¦#! and "−#!antipodes "4a# and "4b# as substrates[ It needs to be determined\ however\ whether the reductase reduces the allylic double bond directly\ or whether some "oxidized# intermediate is involved[ The only other known allylic bond reductase is that of enoyl acyl carrier protein reductase in fatty acid biosynthesis\075\076 which requires the double bond to be in conjugation with a carbonyl group[ The puri_ed dehydrodiconiferyl alcohol 6?07? double bond reductase is thus required\ in order to mechanistically determine how this reduction is achieved[ Additionally\ it needs to be established as to whether the same enzyme catalyzes formation of all of the various {{dihydro|| natural products albeit with di}erent substrate speci_cities\ or if a family of these reductases exists with each catalyzing distinct transformations[

0[14[09[1 Regiospeci_c O!Demethylation at Carbon 2? and Monosaccharide Functionalization In P[ taeda cell suspension cultures\ "2#!dehydrodiconiferyl alcohols "4# can also undergo further metabolism involving regiospeci_c demethylation at C!2?[ As shown in Figure 07\ when "2#! 2 ð8\8?! H1Łdehydrodiconiferyl alcohols "4# were incubated with P[ taeda cell suspension cultures for di}erent time intervals\ both enantiomers were either rapidly demethylated to give demethyl! dehydrodiconiferyl alcohol "030#\ or reduced to a}ord dihydrodehydrodiconiferyl alcohols

Figure 07 Time course of metabolism of "2#!dehydrodiconiferyl alcohols "4a\b#byPinus taeda cell suspension cultures[ Â\"2#!dehydrodiconiferyl alcohols "4a\b#^ R\"2#!dihydrodehydrodiconiferyl alcohols "016a\b#^ ž\"2#!demethyldehydrodiconiferyl alcohols "030a\b#^ r\"2#!cedrusins "029a\b#[ 573 Li`nans] Biosynthesis and Function

"016a\b#[069 In turn\ both metabolites "016a\b# were _nally converted into "2#!demethyl! dihydrodehydrodiconiferyl alcohols "2#!cedrusins "029a\b#^069 the enzymology involved in this demethylation awaits full characterization[

OH HO O MeO OH (141) (Pinus taeda)

Additionally\ these and related derivatives\ whether from dihydrodehydrodiconiferyl alcohol "016#\ dehydrodiconiferyl alcohol "4#\ and guaiacylglycerol 70O03? "dihydro#coniferyl alcohol ethers "5# and "021# are frequently found conjugated to monosaccharides[ They are typically attached to the C!3 phenolic hydroxyl groups with either glucose or rhamnose\ e[g[\ "031# and "032#inPicea abies061 and Pinus silvestris\054\071 and even if the demethylated 2? hydroxyl group is in its free phenolic form[ In other species\ such as Clematis stans\066 the glucose moiety is attached to the 2? hydroxyl position of cedrusin "029# "see "033##\ but not to the 3!hydroxy functionality\ whereas in Licaria chrysophylla "Lauraceae#\051 it is attached to C!8 of dihydrodehydrodiconiferyl alcohol "016# "see "034##[ The signi_cance\ if any\ of these di}erent monosaccharide attachments is unknown[

OR3

OH R1O O 3' MeO OR2 (142) R1 = Glc, R2 = R3 = H (143) R1 = Rha, R2 = R3 = H (Pinus silvestris) (144) R1 = H, R2 = Glc, R3 = H, Clemastanin A (Clematis stans) (145) R1 = R2 = H, R3 = Glc (Licaria chrysophylla)

0[14[09[2 Acylation A few lignans have also been reported in species such as C[ japonica and Corylus sieboldiana which contain acyl\ p!coumaroyl\ or feruloyl moieties conjugated to the lignan skeleton "022#Ð "025#[060\063 These have been noted for both 704? "022#Ð"025# and 707? "035#060 linked lignans\ but nothing has been established about how these trans!esteri_cations and coupling reactions occur[

MeO OAc OAc AcO

OMe OAc (146) (Cryptomeria japonica) Li`nans] Biosynthesis and Function 574 0[14[00 MIXED DIMERS CONTAINING MONOLIGNOLS AND RELATED MONOMERS In addition to the bona _de lignan skeletal types described throughout this chapter\ there are other natural products trivially described as being ~avonolignans\ coumarinolignans\ stilbenolignans\ etc[ These appear to result either from coupling of monolignols\ or in some cases\ lignans\ with other phenolic substances\ such as ~avonoids[ These include\ for example\ the ~avonolignan\ sinaiticin "036# from Verbascum sinaiticum "Scrophulariaceae# leaves077 which appears to result from regio: stereospeci_c coupling of luteolin "037# with p!coumaryl alcohol "21#[ Indeed\ given that the only optical center present in the molecule results from monolignol attachment\ how this coupling is controlled needs to be de_ned[ Related substances such as the optically active hydnowightin "038# are also present in seeds of Hydnocarpus wi`htiana "Flacourtiaceae#\21 again suggesting enzymatic control of coupling in both a regio! and stereospeci_c manner[ More complex ~avonolignans also exist\ such as pseudotsuganol "049#\ which consists of pinoresinol "2# linked to a dihydroquercetin "040# moiety\20\078 this being found in the outer bark of Douglas _r "Pseudotsu`a menziesii#in optically active form "ðaŁD ¦19[1> "c 9[14\ MeOH##[

OH OH O HO O HO O OH OH O

OH O OH O (147) Sinaiticin (148) Luteolin [α]D = Ð15.2¡ (MeOH, c = 0.0033) (Verbascum sinaiticum)

O OH OH OH HO O (149) Hydnowightin O OMe [α]D = +40¡ (MeOH, c = 0.55) (Hydnocarpus wightiana) OH OH O OMe

HO HO O OH OH HO O HO O O OH H OH OH OH HO H OMe OH O O MeO (150) Pseudotsuganol (151) Dihydroquercetin [α]D = +20.2¡ (MeOH, c = 0.25) (Pseudotsuga menziesii) (Pseudotsuga menziesii)

Another interesting group of mixed dimers are the lignanamides\ the jacpaniculines "041# and "042# from the fruits of Jacquemontia paniculata "Convolvulaceae#[089 These presumably result from coupling of E!coniferyl alcohol "27# with feruloyl tyramine "098#\ although nothing can be concluded about how coupling might be carried out\ since the enantiomeric purity of these metabolites has not been described[ On the other hand\ the stilbenolignans\ such as maackoline "043# from Maackia amurensis "Leguminosae# heartwood\080 are apparently racemic suggesting nonspeci_c coupling of the presumed sinapyl alcohol "33# and stilbene precursors[ 575 Li`nans] Biosynthesis and Function

OH OH

O O O O O O N N

MeO OMe MeO OMe O O HO OH HO OH (152) Jacpaniculine (153) Isojacpaniculine (Jacquemontia paniculata) (Jacquemontia paniculata)

OMe OH HO OH

OMe H H

HO OH (154) Maackoline [α]D = 0 (Maackia amurensis)

Other {{mixed dimers|| include "¦#!megacerotonic acid "50# from M[ ~a`ellaris\50Ð52 "−#!cryp! toresinol "02# from C[ japonica "Pinaceae#\06 and the diarylheptanoids\ alnusdiol "044# and maxi! mowicziol A "045# from Betula maximowicziana "Betulaceae# heartwood[081 "¦#!Megacerotonic acid "50# co!occurs with rosmarinic acid "046#\ and its formation "Scheme 08# may result from oxidative coupling as shown\ i[e[\ where generation of the transient biradical species\ derived from "047# gives\ following ring closure and rearomatization\ the 607? linked lignan "50#[ A comparable mechanism may account for the formation of the lignan\ "−#!cryptoresinol "02#[ The diarylheptanoids "044# and "045#\ by contrast\ appear to result from oxidative coupling of two p!coumaryl alcohol "21# molecules via 202? or 20O03? bonds[ Interestingly\ these metabolites contain an additional carbon attached to the 8:8? carbons[ This suggests that the linear diarylheptanoid moiety is _rst formed\ followed by phenoxy radical coupling^ in the latter case\ intramolecular coupling may result from action of a cytochrome P!349 type oxidase\ such as previously noted for "R\S#!berbamunine\ "R\S#! norberbamunine\ and "R\R#!guattegaumerine[040

HO OH OH

3 O 4' 3 3'

OH HO OH OH

(155) (Ð)-(aS, 9S, 9'S)-Alnusdiol (156) (Ð)-rel-(pR, 9S, 9'S)-Maximowicziol A 23 23 [α]D = Ð46.0¡ (MeOH, c = 0.85) [α]D = Ð86.3¡ (MeOH, c = 0.66) (Betula maximowicziana) (Betula maximowicziana)

Finally\ a quite abundant group of {{mixed|| dimers are the so!called cyclobutane lignans found in the Poaceae "Commelini~orae# "61# and "62#57\58 and the Ari~orae "63#[69 These are presumed to be primarily formed via photodimerization of either juxtaposed hydroxycinnamic acids "e[g[\ "18# and "24##\ aldehydes "e[g[\ "20# and "26##\ alcohols "e[g[\ "21# and "27##\ or even allylphenols "e[g[\ "47#Ð"59##[ These molecules are typically attached to the various cell wall fractions of grasses and Li`nans] Biosynthesis and Function 576 grains[ They can apparently be formed by either head!to!head\ head!to!tail\ or tail!to!tail coupling^ all are believed to be optically inactive[

O O CO H O O CO2H O 2 OCO2H H H ¥ ¥

H OH OH OH OH OH OH R OH O O (157) R = OH, Rosmarinic acid (158) R = H, presumed precursor of (+)-megacerotonic acid O O CO2H

OH OH

OH (61)

Scheme 19

0[14[01 LIGNANS AND SESQUILIGNANS] WHAT IS THE RELATIONSHIP TO LIGNIN FORMATION< The view has long been held\61 without rigorous scienti_c proof\ that monolignol "glycoside#s are transported from the cytoplasm into lignifying cell walls where they undergo sequential random coupling to give biopolymeric lignins via transient "oligomeric# lignan formation[ However\ this view must be tempered by the fact that {{random|| coupling of monolignols in vitro never gave an adequate representation of the natural lignin biopolymer"s#\ in terms of\ for example\ frequencies of inter!unit linkages62 "see Chapter 2[07#[ In contrast\ ligni_cation proper has been proposed to occur via end!wise polymerization of the monolignols at discrete points in the cell wall\ this being envisaged to occur along a template viewed to consist of arrays of dirigent protein sites63 or some proteinaceous equivalent\ with the initial lignin strand then replicating via a template mechanism082Ð 084 "see Chapter 2[07 and work by Lewis et al[62#[ Such a process would preclude the formation of transient dimeric and higher oligomeric forms undergoing random coupling\ but would explain all the known features of lignin proper in situ[ On the other hand\ the original random coupling hypothesis was supported in part from analyses of acetone!water extracts from homogenized sapwood and heartwood tissues[ Such preparations were mainly obtained from the Pinaceae\ and were thought at the time to contain {{native or Brauns lignins[||085 Curiously\ while they shared some structural similarities with lignin biopolymers "e[g[\ being coniferyl alcohol "27#!derived#\ they were never proven] "i# to be present in lignifying cell walls\ "ii# to have any structural roles\ and "iii# to be formed via direct monomer polymerization[ Today\ these {{Brauns and native lignins|| appear only to be nonstructural\ nonlignin oligomeric sesquilignans\ which depending upon the plant system involved can apparently exist in a variety of di}erent molecular sizes "see Section 0[14[02[5#[ Their roles appear to be primarily in defense[ For example\ the roots of the herbaceous plant Phryma leptostachya "Phrymaceae# contain a series of insecticidal oligolignans\ such as haedoxan A "048# and its congeners\086 whose formation is clearly under stereospeci_c enzymatic control "Scheme 19#[ It contains a modi_ed 707? furanofuran skeleton\ with an additional C5C2 moiety linked through the 6ý07ý positions as noted for ~avono! lignans such as sinaiticin "036#[ Presumably it is formed via stereospeci_c radicalÐradical coupling of coniferyl alcohol "27# to the preformed dimer "059# or some equivalent thereof\ which then undergoes further metabolism to give haedoxan A "048#[ Such substances clearly would be unable 577 Li`nans] Biosynthesis and Function to undergo further conversion to give lignin biopolymer"s# proper\ or even to be incorporated into the same via a template!replication process[

MeO OH OH O OH Stereospecific + 8 8' OMe radical coupling O O O OMe OH O OMe (38) Coniferyl alcohol (160) Dimer

O H : MeO O: OMe O O OH OMe O O O

O OMe

MeO O O 7" O 8" O 8' HO H OMe 8 OMe O O O

O OMe (159) Haedoxan A 27 [α]D = +125¡ (EtOH-CH2Cl2, c = 0.32) (Phryma leptostachya)

Scheme 20

Other sesquilignans frequently consist of monolignols conjugated to optically active lignan dimers\ such as medioresinol "096#\ secoisolariciresinol "19#\ matairesinol "10#\ dihydrodehydrodiconiferyl alcohol "016#\ and olivil "050#[ These include substances such as hedyotol C "051# from E[ ulmoides087 bark tissue and Hedyotis lawsoniae "Rubiaceae# leaves\72 which can be viewed to result from coupling of medioresinol "096# with coniferyl alcohol "27#[ Another example is dihydrobuddlenol B "052# from Prunus jamasakura "Rosaceae# bark\088 which consists of a 4!methoxydihydro! dehydrodiconiferyl "053# moiety attached to a coniferyl alcohol "27# residue via an 70O03? linkage[ Others include 6?!hydroxylappaol E "054# from hemlock "T[ heterophylla# sapwood\199 consisting of 6?!hydroxymatairesinol "88# linked to coniferyl alcohol "27# via an 70O03? bond\ as well as the lappaols "055# and "056# from the roots of Arctium lappa "Compositae#15\190 and the cerberalignans "057#Ð"069# from Cerbera man`has and Cerbera odollam "Apocynaceae# stem tissue[191Ð193 The lappaols "055# and "056# are primarily matairesinol "10#!derived lignans linked via 704? bonds to coniferyl alcohol "27# residues\ whereas the cerberalignans "057#Ð"069# are mainly Li`nans] Biosynthesis and Function 578

OH OMe OH O O OMe O HO H OMe OH HO OH OMe HO O OMe (161) (Ð)-Olivil HO (162) Hedyotol C 27 26 [α]D = +30.7¡ (MeOH, c = 0.3) [α]D = Ð48.5¡ (MeOH, c = 1.5) OMe

(Cerbera manghas, C. odollam) (Eucommia ulmoides) (Hedyotis lawsoniae) OH OH MeO MeO HO OH OH O HO HO O O OH MeO MeO MeO MeO OMe (163) Dihydrobuddlenol B (164) 5-Methoxydihydrodehydrodiconiferyl alcohol 29 [α]D = Ð28.2¡ (MeOH, c = 0.43) (Prunus jamasakura)

O O MeO 8 MeO 8 O H O O MeO 8 O HO 8' HO 8' HO HO 8'

5' OMe MeO 4' 5' 8" OH MeO OH O 8" O HO 8" OH MeO OH MeO OH OMe HO HO OH (165) (Ð)-7'-Hydroxylappaol E (166) Lappaol A (167) Lappaol C 25 20 20 [α]D = Ð3.7¡ (MeOH) [α]D = Ð17.4¡ (MeOH, c = 1.0) [α]D = Ð55¡ (MeOH, c = 1.0) (Tsuga heterophylla) (Arctium lappa) (Arctium lappa) HO OH OMe MeO MeO OMe

7 O OH OH OH HO 8 8 8''' 5' O OH HO O HO OH OH 5" 8' 8" O HO 8" 7''' OMe 5' MeO HO MeO 5" OMe OH OH OH (169) Cerberalignan B OMe 20 (168) Cerberalignan A [α]D = Ð75.4¡ (MeOH, c = 0.35) 20 [α]D = Ð76¡ (MeOH, c = 0.55) (Cerbera manghas, C. odollam)

(Cerbera manghas, C. odollam) 8' O 5' O OH OMe 8" (170) Cerberalignan D 8 [α] 20 = Ð50.6¡ (MeOH, c = 0.98) OH OH OH D

HO (Cerbera manghas, C. odollam) OMe 589 Li`nans] Biosynthesis and Function "−#!olivil "050#!derived molecules linked head!to!head\ tail!to!tail\ or head!to!tail[ As before\ however\ isolation procedures\ such as for the cerberalignans\ were extremely lengthy and the various lignans isolated were present together with much larger amounts of the presumed precursors\ e[g[\ olivil "059#\ again raising the possibility of artifact formation[ Perhaps one of the most unusual sesquilignan structures proposed is that of herpepentol "060#\ described as present in methanol extracts of grains from Herpetospermum caudi`erum Wall[ "Cucur! bitaceae#[194 Based on its preliminary characterization by FAB MS and 0H NMR spectroscopy\ a coniferyl alcohol "27# derived 704?\ 704?\ 704?\ 707? linked pentamer was proposed[ Although such a substance could only result from endwise coupling "cf[ lignin#\ a more de_nitive study is\ however\ necessary to establish that its proposed structure is correct[

OH O OH OMe OH O OMe O OMe O OMe HO OMe (171) Herpepentol (Herpetospermum caudigerum Wall.)

Thus\ the preponderance of available evidence reveals that "sesqui#lignans have structures which cannot be derived directly via simple free!radical coupling of monolignols\ and therefore lead to lignin formation[ Moreover\ the structural modi_cations typically encountered "e[g[\ methylenedioxy bridge formation\ allylic double bond reduction\ etc[# give substances which "bio#chemically cannot readily undergo further coupling to form high molecular weight lignin biopolymers[ Accordingly\ there is no convincing evidence linking "sesqui#lignan formation to that of the structural lignin biopolymers\ and both pathways must be viewed as being biochemically\ con_gurationally\ temporally\ and spatially distinct "discussed in Section 0[14[02[5#[

0[14[02 PHYSIOLOGICAL ROLES IN PLANTA Although identi_cation of the physiological roles of "oligomeric# lignans is still very much in its infancy\ signi_cant progress in de_ning some of their functions has been made[ The best documented roles appear to be primarily defense related\ namely\ antioxidant\ biocidal\ feeding deterrent\ and allelopathic properties[ As for other systems\ and although this aspect is seldom examined\ a speci_c biological activity can be associated with a particular enantiomeric form[ Using a pharmacological example to illustrate this point\ "−#!trachelogenin "06# inhibits the replication of HIV!0 in vitro\ whereas the corresponding "¦#!antipode is much less e}ective[6 Another signi_cant role of lignans in certain species is in heartwood formation\ since they a}ect color\ durability\ quality\ and texture of the resulting wood] these substances have occasionally been erroneously described as {{abnormal or secondary lignins[|| Other putative roles proposed for the "oligomeric# lignans\ include that of cytokinins044Ð046 and as intermediates in ligni_cation\195 although there are signi_cant counter! arguments to both proposed functions "see Sections 0[14[02[5 and 0[14[01#[

0[14[02[0 Antioxidant Properties Nordihydroguaiaretic acid "061#\ a major constituent of the resinous exudate of the creosote bush "Larrea tridentata#\196 is one of the most powerful antioxidants known\197Ð109 whereas others such as sesamolin "66#\ sesamin "07#\ and sesamolinol "70# from sesame "S[ indicum# seeds display less potent but still striking antioxidant properties[3\87\88 Indeed\ it is perhaps no coincidence that several oil Li`nans] Biosynthesis and Function 580 seed bearing plants\ such as sesame and ~ax\ contain high lignan levels\ which accordingly help stabilize lipid "oil# components against oxidative degradation and onset of rancidity[

OH OH (172) Nordihydroguaiaretic acid

Because of these properties\ several studies have been directed toward de_ning the precise modes of action of lignans in planta and in human applications[ Thus\ nordihydroguaiaretic acid "NDGA# "061# competitively inhibits soybean meal lipoxidase!catalyzed oxidation of sodium linoleate\109\100 an important antioxidant property given that lipoxidase is directly involved in the autooxidation of unsaturated fatty acids during vegetable and seed oil manufacture[ Indeed\ NDGA "061# was a common antioxidant in various foodstu}s until 0861\ when its use was discontinued following indications that it had toxic e}ects on the kidneys[198 It is used instead in nonfood applications\ such as in stabilizing polymers\ rubber\ perfumery oils\ and photographic formulations[

0[14[02[1 Antifungal and Antimicrobial Effects Many lignans have antifungal and antimicrobial properties[ For example\ termilignan "093# and "−#!thannilignan "095#\ from the popular Indian traditional medicinal plant\ T[ bellerica "Combretaceae#\ have potent activities against the fungus Penicillium expansum] the minimum amounts of each lignan required to inhibit fungal growth "0 and 1 mg respectively#\ compared favorably with conventional treatment levels with nystatin "9[4 mg#[026 Additionally\ formation of "−#!matairesinol "10a# and related metabolites\ e[g[\ 6?!hydroxy! matairesinol "88# and a!conidendrin "65#\ is induced in Picea abies upon infection by Fomes annosus\101\102 which\ in turn\ limits further fungal growth[101Ð103 This observation may partly help explain the massive deposition of lignans in the heartwood of western red cedar "T[ plicata#24 which\ in conjunction with tropolones\ helps confer protection to this plant species thereby enabling life spans in excess of 2999 years to be reached[ Other lignans reputed to have antifungal properties include representatives from Podophyllum hexandrum104 and the katsura tree "Cercidiphyllum japonicum\ Cercidiphyllaceae#]105 3?!O!demethyl! dehydropodophyllotoxin "062#\ and picropodophyllone "063# from P[ hexandrum reportedly have antifungal activities against Epidermophyton ~occosum\ Curvularia lunata\ Ni`rospora oryzae\ Micro! sporum canis\ Allescheria boydii\ and Pleurotus ostreatus\ although no quantitative data were given\104 and magnolol "6# from C[ japonicum accumulates in twig cortical tissue in response to Fusarium solani f[ sp[ mori invasion[105 Magnolol "6# is also e}ective against Asper`illus ni`er and Tricophyton menta`rophytes with minimum inhibitory concentrations "MIC# of 29 and 1[4 mgml−0\ respectively\ which compares well with the control using amphotericin B "MIC 29 and 04 mgml−0#[106

OH O H O H O O O O H O O H O

MeO OMe MeO OMe OH OMe (173) 4'ÐOÐDemethyldehydropodophyllotoxin (174) Picropodophyllone 581 Li`nans] Biosynthesis and Function There have also been a limited number of studies examining both regio! and enantiospeci_c conversions of lignans in response to exposure to various fungi[ For example\ "2#!eudesmins "72#\ when incubated with Asperìllus nièr\ are converted into both pinoresinol monomethyl ether "78# 18 18 "ðaŁD −01[7>\ enantiomeric excess 28[2)# and pinoresinol "2#"ðaŁD −46[5>\ enantiomeric excess 099)#\ where the "−#!antipode was more rapidly demethylated than its "¦#!counterpart[107 Why this organism preferentially metabolizes one particular enantiomer may be of signi_cance in plantÐ fungus interactions[ This may also be particularly important in lignin biodegradation studies\ which most commonly use lignans rather than lignins for their assays[ Unfortunately\ none of the lignin! degradation studies have examined whether conversions with lignans are enantiospeci_c[ A number of other lignans are also regiospeci_cally demethylated at the para position by A[ nièr\ notably "¦#!magnolin "73#\ "¦#!epimagnolin A "064#\ "¦#!veraguensin "065#\ "¦#!galbelgin "066#\ and galgravin "067#\ but not "¦#!yangambin "74#[108Ð110 This fungus can also de!ethylate diethyl pinoresinol "068# and its monoethyl derivative "079# to give pinoresinol "2#\ but has no e}ect on dipropyl "070# and dibutyl "071# analogues[111

OMe

O OMe H H MeO O MeO OMe O MeO OMe MeO OMe (175) (+)-Epimagnolin (176) (+)-Veraguensin

MeO OMe MeO OMe O O

MeO OMe MeO OMe (177) (+)-Galbelgin (178) Galgravin

OR2

O OMe

R1O OMe (179) R1 = R2 = Et, (+)-Diethyl pinoresinol (180) R1 = Et, R2 = H, (+)-Monoethyl pinoresinol (181) R1 = R2 = nPr, (+)-Dipropyl pinoresinol (182) R1 = R2 = nBu, (+)-Dibutyl pinoresinol

Interestingly\ and while the signi_cance is again unknown\ Fusarium solani enantiospeci_cally converts D7?!hydroxy!2\2?dimethoxy!6!oxo!7!O!3?!lignan "072#*when incubated in the presence of the ketone "073#*into the threo:erythro lignans "018# in a ratio of 1]2 "Scheme 10#[ For both threo and erythro forms\ only one enantiomer was formed\ revealing that the reductive step was fully enantiospeci_c[056 Lignans also have antibacterial properties] magnolol "6# and honokiol "074# inhibit Staphylococcus aureus\ Bacillus subtilis\ and Mycobacterium sme`matis bacterial growth with a MIC of 4Ð09 mgml−0\ which is comparable to or better than the activity of streptomycin sulfate "MIC 09\ 09\ and 1[4 mgml−0\ respectively#106\ and nordihydroguaiaretic acid "061# is e}ective against salmonella\ penicillium\ M[ pyro`enes\ and Saccharomyces cerevisiae[198 Extracts from the aril tissue of Myristica Li`nans] Biosynthesis and Function 582

HO CHO

O O OMe

OMe O HO O OH O (184) OMe OMe

OMe OMe OH OH (183) (129)

Scheme 21

fra`rans "mace# are also used in Sri Lanka for dental caries prevention "antiplaque formation#\ with dehydrodiisoeugenol "licarin A# "46# and 4?!methoxydehydrodiisoeugenol "075# being characterized as the major antibacterial principles[ Both inhibit the growth of Streptococcus mutans at con! centrations of 01[4 mgml−0[112

OH MeO

HO OH O MeO OMe

(185) Honokiol (186) 5-Methoxydehydrodiisoeugenol

0[14[02[2 Insecticides\ Nematocides\ Antifeedants\ and Poisons "¦#!Haedoxan A "048#\ isolated from the roots of the herbaceous perennial plant\ Phryma leptostachya\ is perhaps the best known insecticidal lignan[086\113Ð115 In combination with piperonyl butoxide "a synergist#\ it has excellent insecticidal activity\ when administered orally to several 114 lepidopterous insect larvae and house~ies\ e[g[\ Musca domestica\LD49 9[14 ng per ~y\ this being comparable to that of commercial synthetic pyrethroids[ Its physiological e}ect results in muscle relaxation\ feeding cessation\ general paralysis\ and death\ thereby causing similar e}ects to the insect neurotoxins\ nereistoxin\ ryanodine\ and reserpine[086 Another insecticidal lignan is the 7\4?!linked\ licarin B "076#\ isolated from Myristica fra`rans\ which is e}ective against silkworm "Bombyx mori# fourth instar larvae "at 299 ppm in the diet#\ with death occurring 2 days or so after treatment[116 Additionally\ the lignans "077#\ "078#\ and magnolol "6#\ isolated from Ma`nolia vir`iniana "Magnoliaceae#\ cause 099) mortality to mosquito "Aedes ae`ypti# larvae at con! centrations of ½09 ppm within 1 hours\ this being comparable to valinomicin treatment as a control[117

OH O OH O HO O O OMe OMe

(187) Licarin B (188) (189)

Growth inhibitory properties of lignans have also been described] "¦#!epimagnolin A "064#\ isolated from the ~ower buds of Ma`nolia far`esii\ inhibits the growth of D[ melano`aster larvae118 at concentrations greater than 0 mg ml−0\ whereas licarin A "46#\ "−#!machilusin "089#\ and lignans 583 Li`nans] Biosynthesis and Function "080# and "081# from the leaves of Machilus japonica function by inhibiting Spodoptera litura larval 129 growth when added to their diets "EC49 9[19\ 9[08\ 9[02\ and 9[13) w:w\ respectively#[ "¦#!Sesamin "07# and "¦#!sesamolin "66# from S[ indicum also synergistically act with natural juvenile hormone to prevent metamorphosis in the milkweed bug "Oncopeltus fasciatus# at amounts of 09 and 0 mg\ respectively[120 Moreover\ "¦#!sesamin "07# and epi!sesamin "082# function with pyrethrum insecticides\ by inhibiting oxidative degradation "cytochrome P349 oxygenase system# in the gut of the ingesting organism[4\121

MeO

O OMe MeO O O O OMe OMe (190) (Ð)-Machilusin (191) O O

OMe H O H O O OMe (192) O O (193) Epi-sesamin

Lignans confer protection against nematodes[122\123 For example\ "−#!matairesinol "10a# and "−#! bursehernin "083#\ at concentrations of ½49 mgml−0\ inhibit the hatching of potato cyst nematodes\ Globodera pallida and G[ rostochiensis\ by 44) and 69)\ respectively\ when compared with controls −0 using ZnSO3 and ethanol] the hatching inhibitory dose "05[31 mgml # for bursehernin "083# reduced hatching by 49) over a 1!week period[ The presence of the methylenedioxy bridge seems to play an important role\ since when replaced with either a methoxyl\ one hydroxyl\ or two hydroxyl groups\ the inhibitory activity was greatly reduced[ Interestingly\ lariciresinol 8!O!b!D!glucoside "69#\ isolated from the roots and stolons of potatoes\ accumulates in response to infection with G[ rostochiensis\124 perhaps also suggesting a nematocidal role[

O O O O

OMe OMe (194) Bursehernin

Several lignans display antifeedant and:or toxicity e}ects[ "−#!Yatein "084# has antifeedant properties when added to the diets of adult granary weevil beetles "Sitophilus `ranarius# and confused ~our beetle "Tribolium confusum# with an estimated total coe.cient of deterrency of 078 and 047\ respectively\ where values × 199 indicate very good feeding!deterrent activity[125 "¦#!Eudesmin "72#:"¦#!epi!eudesmin "085# from Parabenzoin praecox\ and "−#!piperenone "086# from Piper futo! kadsura\ have antifeedant activities "89Ð099)# against S[ litura larvae when provided at con! centrations of 9[94)\ 0[9)\ and 9[994) in the diet[126\127 Justicidins A "087# and B "088# from Justicia hayatai var[ decumbens\ on the other hand\ show strong toxicity against Oryzias latipes at levels comparable to that of rotenone and 09 times higher than that of pentachlorophenol[ J[ hayatai has been used for many centuries as a _sh!poison by the natives of the Pescadores "Pung Fu islands# of Taiwan[128 Li`nans] Biosynthesis and Function 584

OMe O OMe O O O O H H O MeO OMe

OMe MeO OMe (195) Yatein (196) Epi-eudesmin

O R MeO MeO O O OMe MeO O

MeO O MeO O (197) Piperenone (198) R = OMe, Justicidin A (199) R = H, Justicidin B

The lignanamides\ "2#!grossamide "005#\ "2#!demethylgrossamide "006#\ "−#!cannabisin B "003#\ and "−#!cannabisin D "004#\ isolated from X[ aethiopica also display antifeedant properties at 4999 ppm against subterranean termite "Reticulitermes speratus# workers] index values of 0[80\ 18[38\ 6[09\ and 01[82\ respectively\ were obtained\ where ³ 19 indicates signi_cant feeding!deterrent activity[032

0[14[02[3 Allelopathy Various lignans have powerful allelopathic properties[ For example\ nordihydroguaiaretic acid "061#\ when supplied at concentrations of ½19 mgl−0\ is able to dramatically reduce the seedling root growth of barnyard grass\ green foxtail\ perennial ryegrass\ annual ryegrass\ red millet\ lambsquarter\ lettuce\ and alfalfa\ as well as the hypocotyl growth of lettuce and green foxtail[139 Additionally\ the monoepoxylignanolide "199#ofAe`ilops ovata is reputedly a unique germination inhibitor of Lactuca sativa "lettuce# achenes\ this e}ect being greater in the light than under darkness[130 Arctiin "81# from Arctium lappa inhibits the germination of 00 out of 01 di}erent plant species tested at concentrations of ½4 mg ml−0\ and the levels of arctiin "81# parallel that of the annual rhythm of germination[131 The furofurans\ fargesin "190# and sesamin "07#\ are germination inhibitors of peanut "Arachis hypo`aea# seeds "lipid!storing seeds#\ but not of rice "Oryza sativa# seeds "carbohydrate!storing seeds#\132 where the investigators suggested that this e}ect might be on processes:enzymes controlling lipid metabolism[ Interestingly\ in the same studies\ eudesmin "72#\ which lacks a methylenedioxy group\ was much less active\ suggesting that the methylenedioxy group is needed for biological activity[

0[14[02[4 Cytokinin!like Activities Certain lignans have been implicated to function as cytokinins during plant growth and develop! ment\ although they are only e}ective at very high concentrations[ For example\ a cytokinin!like 585 Li`nans] Biosynthesis and Function

OMe OMe O OMe O H OH O H O

HO OMe O O (200) (201) Fargesin function has been proposed for "2#!dehydrodiconiferyl alcohol 3!O!glucosides "014a\b#\ since they can stimulate cell division of tobacco "N[ tabacum# cells and replace cytokinin in pith and callus cultures]044Ð046 both "¦#! and "−#!dehydrodiconiferyl alcohol 3!O!glucosides "014a# and "014b# stimulated pith growth\ at concentrations of ½09 mM\ in a manner comparable to that of the cytokinin\ zeatin riboside "9[0 mM#[ On the other hand\ they apparently did not stimulate shoot formation from leaf explants as normally observed for cytokinins[044 These investigators also pro! posed that the glucosides were being mobilized from their roles as cell!wall components\ even though this could not be the case since they were extracted from the callus cultures by methanol:water extraction[ That is\ although their subcellular origins were not determined\ they could not have been cell!wall constituents based on this solubilization property[ Moreover\ these data must be viewed as only a tentative indication of any cytokinin role in vivo\ and more extensive studies are required to prove that this is indeed a true function[

0[14[02[5 Constitutive and Inducible "Oligomeric# Lignan Deposition and Nonstructural Infusions\ {{Abnormal|| and {{Stress|| Lignins The physiological roles of lignans discussed thus far are those of antioxidant\ biocidal\ allelopathic\ and antifeedant agents\ as well as a putative role as cytokinins[ These properties\ in turn\ lead to the question of what are the factors controlling their induction:constitutive formation in planta< However\ with the appropriate gene76\89\000 and promoter sequences "data not shown# in hand\ together with their proteins and antibodies\ meaningful experiments can be undertaken to de_ne how their formation is regulated\ and the temporal and spatial nature of expression of the biosyn! thetic pathways involved[ What is known about sites of lignan accumulation is both rudimentary and variable\ with evidence from di}erent studies perhaps pointing to di}erent locations[ One study suggests that 4! methoxypodophyllotoxin "099# is present in the vacuolar compartments of Linum album cell sus! pension cultures\ this being based on crude subcellular "organelle# fractionation studies[26 That it is supposedly present in the vacuoles might also suggest a similar location for the constitutively formed insecticidal lignan\ haedoxan A "048#\ in the roots of P[ leptostachya\ and the cytotoxin\ podophyllotoxin "12#\ in the rhizomes of P[ peltatum:P[ hexandrum[ In seed tissues\ such as sesame and ~ax\ nothing is de_nitively known about their lignan subcellular locations\ a situation which also holds for lignans in ~owers\ fruits\ leaves\ and bark[ Lignan deposition in sapwood and heartwood tissues of certain woody plants has been a matter of particular interest\ given the long!standing confusion surrounding the nature of various heartwood metabolites and whether they are lignins\ lignans\ or oligomeric lignans "see Section 0[14[01#^ this\ in turn\ has led to the use of lax terminology to describe such constituents\ e[g[\ as {{abnormal and secondary|| lignins[ Heartwood formation itself is initiated\ at some undetermined point\ in the center "pith# region of mature preligni_ed secondary xylem wood[ What initiates or induces its formation is unknown\ although the metabolic composition "lignans\ ~avonoids\ alkaloids\ etc[# can vary extensively with the species[ Nevertheless\ it is this phenomenon that provides the various woody types\ with these di}ering in terms of color\ quality\ durability\ and rot resistance\ e[g[\ the black color of ebony wood and the reddish!brown color of western red cedar are due to their distinctive heartwood Li`nans] Biosynthesis and Function 586 metabolites\ whereas the whitish!yellow of spruce results from the near absence of any heartwood metabolites[ Heartwood!forming substances are considered to be initially formed and released from specialized ray parenchyma cells\ with these substances further infusing into neighboring tracheids and:or _bers:vessels "Figure 08#[ As proposed by Chattaway nearly 49 years ago\133\134 they are exuded through parenchyma cells via pit apertures into the lumen of adjacent dead\ preligni_ed\ cells and then di}use into neighboring\ preligni_ed cells to ultimately a}ord the heartwood tissue[ In agreement with this contention\ Hergert|s analysis of western red cedar and western hemlock constituents also led to the conclusion that heartwood!forming substances accumulated in ray parenchyma cells\ prior to becoming insoluble infusions:deposits in tracheid cells[18 Indeed\ for these reasons\ Hergert cautioned that analytical results obtained from the analysis of wood samples must specify the physiological conditions and:or tissue\ e[g[\ whether sapwood\ heartwood\ com! pression wood\ diseased wood\ etc[ Unfortunately\ most investigations treat woody tissue as if it were homogeneous[

Figure 08 Secretion of heartwood constituents by ray parenchyma cells into the lumen of neighboring cells appears to occur through pit apertures[ After Chattaway[133

Heartwood metabolites are frequently\ but erroneously\ also described as {{extractives\|| based on the fact that a portion can be removed by aqueous:organic solvent treatment[ It is seldom acknowl! edged\ however\ that only a proportion of these metabolites is solubilized\ with the remaining often requiring harsher conditions\ e[g[\ as commonly employed for lignin dissolution[ To a lesser extent\ comparable substances to those present in heartwood can also be formed in sapwood\ in response to biological challenges\ such as insect attack[ Unfortunately\ imperfect characterization of such heartwood:sapwood metabolites\ particularly when lignan derived\ has led to their descriptions as {{abnormal|| lignins\ {{secondary|| lignins\ and {{Brauns native|| lignins[ These substances are\ however\ not lignins\ but instead constitute "see Section 0[14[01# a fundamentally distinct biochemical class of nonstructural\ nonlignin metabolites which can be produced in a range of sizes and form insoluble deposits during heartwood formation\ i[e[\ they di}er from lignins in terms of temporal and spatial deposition\ con_gurations of the molecules\ postcoupling modi_cations\ and physiological function[ In western red cedar\ for exam! ple\ the lignan!derived components can range from monomers\ such as plicatic acid "64# and plicatin "191# to oligomers "MW ½09 999#\ with a portion only being removed under conditions required for lignin removal[16Ð18\25 Another example is western hemlock heartwood\ the lumen of whose tracheid cells can contain either 6?!hydroxymatairesinol "88#ora!conidendrin "65#[135 This het! erogeneity is particularly interesting\ since it suggests that certain parenchyma cells may be involved in formation of speci_c metabolites\ regardless of the fact that complex mixtures may ultimately result in the developed heartwood via the infusion process previously discussed[133\134 587 Li`nans] Biosynthesis and Function

OH MeO O HO OH O

HO OMe OH (202) (Ð)-Plicatin

As described in Section 0[14[01\ other nonlignin\ nonstructural metabolites are that of the {{Brauns native|| lignins085 and {{abnormal|| lignins136 present in both Pinaceae heartwood and sapwood "although typically by 09!fold less in the latter#[ These substances\ e[g[\ in loblolly pine "Pinus taeda#\ are primarily dehydrodiconiferyl alcohol "4a\b# derived\ and result from lignan modi_cations via allylic double bond reduction\ demethylation\ and phenylcoumaran ring opening[060Ð062 As before\ they are released into the preligni_ed sapwood via specialized cells\ and have chemical structures which preclude them from being able to undergo polymerization to give lignin biopolymers "see Section 0[14[01 and Chapter 2[07#[ The term\ {{stress|| lignins\ has also been introduced to describe inducible {{lignin!like|| responses\ but again with no explicit biochemical explanation as to what it actually meant[ For example\ P[ taeda cell suspension cultures "and those of other plants in the Pinaceae# can be induced\ at high sucrose concentrations\ to form a lignin!like {{extracellular|| precipitate[057 Detailed analysis of these substances revealed\ however\ that this inducible response resulted from lignan coupling as for the {{abnormal|| lignins\ and as such they more closely resemble constituents formed during heartwood deposition which are formed by a distinct biochemical pathway to that of the lignins[ This\ however\ again underscores the necessity to use molecular probes to comprehensively distinguish\ both temporally and spatially\ between lignin and "oligomeric# lignan formation and deposition[

0[14[03 ROLES IN HUMAN NUTRITION:HEALTH PROTECTION AND DISEASE TREATMENT Lignans have long held considerable importance in medicine\ human health\ and nutrition\ and a brief description of some of their most signi_cant applications is summarized below[

0[14[03[0 Nutrition:Health and Protection against Onset of Breast and Prostate Cancers] Secoisolariciresinol\ Matairesinol\ and Sesamin Dietary lignans\ e[g[\ secoisolariciresinol "19# and matairesinol "10#\ have signi_cant roles in conferring health protection\ particularly against the onset of breast and prostate cancers[ During digestion\ they are metabolized into the {{mammalian|| lignans\ enterodiol "192# and enterolactone "193#\ these being _rst detected in the urine of female rats and humans[137\138 Interestingly\ excretion of these lignans had a cyclic pattern during the menstrual cycle\ which reached a maximum during the luteal phase[ It has also been shown with rats that the levels of enterodiol "192# increase substantially when ~axseed was provided\ due to metabolism of secoisolariciresinol diglucoside "87#[149

HO HO OH O OH

OH OH (203) Enterodiol (204) Enterolactone Li`nans] Biosynthesis and Function 588 Recognition of the health!protection e}ects of dietary lignans began with observations of sig! ni_cant metabolic pro_le di}erences in the urinary excretions from individuals at low risk for breast:prostate cancers\ and those at high risk or who had contracted these cancers[ Low breast cancer risk Finnish women\ for example\ had high levels of mammalian lignan!derived metabolites\ enterodiol "192# and enterolactone "193#\ in their body ~uids "urine\ plasma\ and bile#\ whereas those at high risk did not "³ 4) of total#[140Ð142 This was subsequently recognized to be a form of chemoprotection which was ultimately correlated with a vegetarian!like diet "grains\ _bers\ seeds\ and berries# rich in the lignans\ secoisolariciresinol "19# and matairesinol "10#[ Their subsequent conversion into the protective {{mammalian|| lignans\ enterodiol "192# and enterolactone "193#\ occurs in the gut143\144 via loss of the hydroxyl functionalities at C!3 and C!3? and demethylation at C!2 and C!2?[ Scheme 11 shows a possible biochemical pathway[

MeO MeO OH O OH HO HO O

OMe OMe OH OH (20) Secoisolariciresinol (21) Matairesinol

MeO MeO OH O OH

OMe OMe

HO HO OH O OH

OH OH (203) Enterodiol (204) Enterolactone

Scheme 22

Dietary lignans "as well as iso~avonoids# impart chemoprotective e}ects due to their antioxidant\3\87\88 weak oestrogenic:antioestrogenic\145Ð147 anti!aromatase\148\159 and anticarcinogenic: antitumor150Ð154 properties\ thereby protecting against the initiation of various sex hormone!induced cancers[ Their importance can be illustrated using as an example the sex hormone binding globulin "SHBG#[ This binds circa 49) of circulating testosterone in men\ and 79) of the oestrogen in women147 and thus the availability of sex hormone to target cells is greatly a}ected by changes in both its concentration and:or binding properties[ Postmenopausal women excreting large amounts of mammalian lignans have higher levels of SHBG than omnivores or breast cancer patients\154 and other studies have shown that mammalian lignans and iso~avonoids interact with SHBG in a dose! dependent manner\ with enterolactone "193# × equol "194# × genistein "195# for displacing estradiol "196#\ and equol "194# × enterolactone "193# or enterodiol "192# × genistein "195# for testosterone "197# displacement[147\155 These e}ects occur at levels "4Ð49 mM# which correspond well with the 699 Li`nans] Biosynthesis and Function concentrations of plant!derived dietary diphenols in body ~uids\ following ingestion of vegetarian and soy diets\ thereby suggesting that sex!hormone binding is modulated by their presence[

HO O HO O

OH O OH OH (205) Equol (206) Genistein

HO OH

HH OH O (207) Estradiol (208) Testosterone

There is also chemoprotection against the onset of tumors[ SpragueÐDawley female rats\ pre! treated with the "pro#carcinogen dimethylbenzanthracene\ had reduced incidence rates "26) reduction# and tumor masses "35) reduction in mammary tissues#150 when rats were administered a diet containing secoisolariciresinol diglucoside "87#\ and the number of valid putative preneo! plastic markers for colon carcinogenesis in male rats also declined[152 Treatment with ~axseed or the ~ax lignan\ secoisolariciresinol diglucoside "87#\ also signi_cantly reduced epithelial cell proliferation\ as well as the number of aberrant crypts and aberrant crypt foci\ viewed to be early indicators of colon cancer risk[152 In this regard\ it is noteworthy that both Japanese and Caucasian men have comparable numbers of precancerous colonic aberrations^ however\ in Japanese men\ somehow these do not become cancerous to the same extent\ an observation which is viewed as a dietary consequence[156 Such dietary di}erences may also help explain the di.culties in inducing carcinogenesis in primates\ even when procarcinogens are added to the diet[ For example\ admin! istering oestrogens with benzpyrene:dibenzanthracene to rhesus monkeys did not result in car! cinogenesis\ even after 7 years[153 This e}ect is presumed to be due to the fact that primates have diets which result in the massive accumulation and excretion of lignan "as well as iso~avone# diphenolics[ Flaxseed is the richest source of mammalian lignan precursors\ containing levels 64Ð799 higher than any other plant food\ and is being widely investigated for its cancer protective e}ects[ Incu! bation of its most abundant lignan\ "¦#!secoisolariciresinol diglucoside "87#\ with cultured human fecal micro~ora in vitro\ suggested the metabolic pathway "Scheme 11# for its conversion into the mammalian lignans] that is\ intestinal bacteria hydrolyzed the sugar moiety to release seco! isolariciresinol "19#\ this being presumably followed by dehydroxylation and demethylation to give the mammalian lignan enterodiol "192#\ which was oxidized to enterolactone "193#[ As indicated in Scheme 11\ enterolactone "193# is also considered to result from catabolism of the plant lignan\ matairesinol "10#[157 Once formed\ both mammalian lignans undergo enterohepatic circulation\158 where a good correlation exists with their presence and the reduced incidence rates of hormone! related cancers[ Chemoprotection can also be correlated with antioxidant abilities\ e[g[\ of secoisolariciresinol diglucoside "87# as a radical scavenger[169 For example\ hydrogen peroxide\ when subjected to photolysis under ultraviolet light in the presence of salicylic acid\ is involved in the formation of the OH= adduct products\ 1\2!dihydroxybenzoic acid "DHBA# and 1\4!DHBA[ When seco! isolariciresinol diglucoside "87# was present\ however\ a concentration!dependent decrease in the formation of 1\2!DHBA and 1\4!DHBA occurred due to scavenging of OH= radicals by seco! isolariciresinol diglucoside "87#[ On a somewhat related topic\ dietary sesamin "07# also has the e}ect in vivo of elevating levels of the antioxidant\ g!tocopherol\ in rat plasma and liver\ leading to the suggestion that a sesame!rich diet increases availability of antioxidants "vitamin E# in the body[ This\ in turn\ decreases the risk of a number of diseases directly related to free!radical formation\ such as more rapid onset of aging^160 sesame lignans can also cause an increase in vitamin E activity in rats fed a low a!tocopherol diet[161 Li`nans] Biosynthesis and Function 690 0[14[03[1 Antitumor Properties] Podophyllotoxin and other 707? Lignans There are essentially only a handful of plant natural products used in medicine today in cancer treatment\ of which one is podophyllotoxin "12# from Podophyllum peltatum and Podophyllum hexandrum[ Its pharmacological usage dates back many centuries\ when may apple "P[ hexandrum# alcoholic extracts\ obtained from rhizomes and roots\ were employed _rst as a poison and later\ in smaller doses\ for treatment of various pathological conditions[162 The cytotoxic e}ect of these extracts was subsequently found to be due to podophyllotoxin "12#\ which led to its use as an antitumor agent against various malignancies[ It was later shown that podophyllotoxin "12# was readily taken up by the cells due to its small size and hydrophobicity\ with tubulin binding "at a di}erent site to that occupied by the Vinca rosea alkaloids# and microtubular assembly inhibition arresting cells in mitosis^163\164 this occurred in a manner more rapid and reversible than colchicine[ Its action leads to cytoskeletal arrest of cell division and ultimately cell death[ In spite of its antitumor promise\ the clinical applications of direct administration of podo! phyllotoxin "12# were greatly compromised by severe "gastrointestinal# toxicity experienced by those under treatment[ Accordingly\ a signi_cant e}ort was launched to identify means whereby the drug could be delivered with reduced cytotoxicity[ This led to the development\ and subsequent wide! spread application\ of its semisynthetic derivatives\ etoposide "090# and teniposide "091#\165\166 which are used "alone or in conjunction with other drugs\ e[g[\ cisplatin# for treatment of Hodgkin|s lymphomas\ non!Hodgkin|s lymphomas\ small cell lung cancers\ and acute leukemias[167Ð179 The semisynthetic derivatives "090# and "091#\ however\ were found to di}er in their mechanism of action[ The added sugar moieties prevented tubulin interactions from occurring\ and thus micro! tubular assembly was not inhibited[ The antitumor action was\ instead\ a consequence of an ability to form stable tertiary complexes with topoisomerase II and its substrate DNA leading to formation of numerous double!stranded DNA breaks[ In turn\ this results in large DNA fractures\ thereby 170 arresting cells in their life cycle at the G1 phase\ and ultimately causing cell death[ At present\ etoposide phosphate "198#\ the phosphorylated form of etoposide "090#\ is undergoing clinical tests\ since its application may be more convenient due to its increased water solubility[171\172

Me O O O HO HO O O 8' O

O 8 O

MeO OMe

OPO3Na2 (209) Etoposide phosphate

Other lignans have promising anticancer properties] "−#!steganacin "14# and "−#!steganangin "109#\ isolated from S[ araliacea stem bark and wood\ exhibit antileukemia activities both against the in vivo murine P!277 lymphocytic leukemia test system and in vitro against cells derived from a human carcinoma of the nasopharynx cell culture[037 It is thought that this antimitotic activity is through an e}ect on spindle microtubules\ as for podophyllotoxin "12#\ with the chirality about the pivotal biphenyl bond and the orientation of the lactone carbonyl being essential for antitumor activity[173 Epi!steganangin "100# and steganoate B "101# also have cytotoxic properties against 00 di}erent human tumor cell lines\174 and "−#!burseran "102# from Bursera microphylla "Burseraceae# displays antitumor properties against human epidermoid carcinoma of the nasopharynx cell culture[175\176 "¦#!Wikstromol "56#\ from Wikstroemia foetida var[ oahuensis Gray "Thymeleaceae# is also active against the P!277 lymphocytic leukemia test system[177 Finally\ phyllanthin "103# and hypophyllanthin "104#\ from Phyllanthus amarus Schum[ + Thonn[ "Euphorbiaceae#\ enhance cytotoxic responses mediated by vinblastine in the multidrug!resistant KB cell line[ However\ alone they had no signi_cant cytotoxic activity with a large number of mammalian cells[178 691 Li`nans] Biosynthesis and Function

O O MeO O O O O O OMe O O O O O O O O O O O

MeO MeO MeO OMe MeO OMe OMe MeO OMe MeO OMe MeO (210) (Ð)-Steganangin (211) (Ð)-Episteganangin (212) (Ð)-Steganoate B (213) Burseran

MeO MeO OMe OMe OMe OMe MeO O O

OMe OMe OMe OMe (214) Phyllanthin (215) Hypophyllanthin

0[14[03[2 Hepatotoxic Preventive Effects Schizandra chinensis fruit is an important component of various traditional Asian medicines[ Its {{kita!gomisi|| extract is used as an antitusive and tonic\038 whereas the {{Sheng Mai San|| formulation is employed in the treatment of coronary heart disease treatment[189 The fruits have also been used in Japan and East Asia for the treatment of elevated serum aminotransferase activity in acute hepatitis[180 Schizandra fruit contains signi_cant levels of the 707?\1!1? linked lignans\ such as gomisin A "010#[181 This lignan appears to have an excellent ability in protecting the liver from a variety of liver!damaging agents\ such as the hepatotoxic compounds\ CCl3\ galactosamine\ and lipopoly! saccharides[180\182\183 This protection has been correlated with several enzymatic processes\ e[g[\ inhibition of leukotriene biosynthesis preventing arachidonic acid release\184 and prevention of acetaminophen!induced liver injury in rats by inhibiting lipid peroxidation[180 Prevention or limi! tation of acetaminophen!caused intoxication by gomisin A "010# is considered to be due to the reduction of aminotransferase activity in serum and suppression of lipoperoxide accumulation in liver[180 Liver regeneration\ following partial hepatectomy\ is also stimulated by gomisin A "010#\ and this is thought to occur via stimulation of ornithine decarboxylase activity leading to putrescine and spermidine accumulation "polyamines play an important biochemical role in liver regen! eration#185 as well as DNA and RNA biosynthesis[186 Gomisin A "010# also has an ability to inhibit 2?!methyl!3!dimethylamino!azobenzene induced liver carcinogenesis\187\188 as well as limiting muscular damage induced by excessive exercise[299

0[14[03[3 Antiviral Properties Many lignans have been demonstrated to exhibit quite potent antiviral properties] podo! phyllotoxin "12# and "−#!a!peltatin "105# prevent development of murine cytomegalovirus plaques in mouse 2T2!L0 cells\ as demonstrated in vitro by reduction of plaque numbers by ½49) at a concentration of 09 ng ml−0[290 Rhinacanthin E "106# and rhinacanthin F "107#\ from the medicinal plant Rhinacanthus nasutus "Acanthaceae#\ also have antiviral activities against the in~uenza type

A virus[ Using the hemadsorption inhibition assay\ these lignans have EC49 values of 0[6 and ³ 9[83 mgml−0\ whereas with a cytopathic e}ect assay dosage levels were 6[3 and 2[0 mgml−0\ respectively[ For both assays\ control values with amantadine and ribavirin were 9[943 and 2[6 mgml−0\ respectively[291 Li`nans] Biosynthesis and Function 692

OH O CO2Me O CO2Me O O O O O CO2Me CO2Me O

MeO O MeO O MeO OMe O O OH (216) (Ð)-α-Peltatin (217) Rhinacanthin E (218) Rhinacanthin F

A number of lignans inhibit replication of the human immunode_ciency virus "HIV#\ albeit with di}erent modes of action[ "−#!Arctigenin "11a# and "−#!trachelogenin "06# inhibit HIV!0 replication in infected human cell systems\6 with "−#!arctigenin "11a# suppressing integration of proviral DNA into the cellular DNA genome\292 whereas it was inactive with puri_ed HIV!0 integrase[293 2\2?! Demethylarctigenin "108# "a catechol analogue#\ on the other hand\ gave a strong inhibition of HIV!0 integrase[293

HO O HO O

OH OH (219) 3,3'-Demethylarctigenin

Antiviral e}ects have also been noted for constituents from the creosote bush\5\294 a plant widely used in traditional medicine among the indigenous people of America for digestive disorders\ rheumatism\ venereal diseases\ and sores[ One of its lignans\ 2?!O!methylnordihydroguaiaretic acid 5 "119#\ inhibits HIV Tat!regulated transactivation in vivo "EC49 14mM#\ and it is thought that this occurs by interrupting not only the life cycle of wild!type HIV\ but also of reverse transcriptase or protease mutant viruses[294 Other tetrahydronaphthalene lignan analogues have been tested as potent inhibitors of HIV!0^ of these\ the most e}ective is compound "110#\ which has an ED49 of 9[7 mM with an IC49 of 47 mM[ It functions as a noncompetitive inhibitor of HIV!0 reverse tran! scriptase with respect to both template!primer and substrate "dGTP#\295 as does phyllamycin B "111# and retrojusticidin B "112# from Phyllanthus myrtifolius "Euphorbiaceae^ IC49 2[4 and 4[4 mM\ respectively# with respect to template primer and triphosphate substrate[296

MeO MeO MeO O OMe S O H HO MeO MeO O O MeO O

OH MeO OMe O MeO OH OMe O OMe

(220) 3'-O-Methylnordihydro- (221) (222) Phyllamycin B (223) Retrojusticidin B guaiaretic acid

Of the Schizandra lignans\ examined for anti!HIV activity\ "−#!gomisin J "113a# displays bene_cial e}ects\ whereas gomisins A "010#\ D "114#\E"115#\ and N "116#\ deoxyschizandrin "117#\ and "¦#! gomisin J "113b# do not[297 Interestingly\ the synthetic bromine analogue "118# of "−#!gomisin J was 22!fold more e}ective than "−#!gomisin J "113a# itself[ Studies of its action suggested it to be both a noncompetitive inhibitor of HIV!0 reverse transcriptase\ and a mixed "noncompetitive and uncompetitive# inhibitor with respect to the primer!template[297 The bromo derivative "118# was also e}ective against 2?!azido!2?!deoxythymidine "AZT# resistant HIV!0\ as well as synergistically 693 Li`nans] Biosynthesis and Function acting with AZT[ Finally\ anolignan A "129# and anolignan B "094#\ from Ano`eissus acuminata "Combretaceae#\ synergistically inhibit HIV!0 reverse transcriptase\298 whereas interiotherin A "120# and schisantherin D "121#\ isolated from Kadsura interior "Schizandraceae#\ inhibit HIV replication 209 with EC49 values of 5[0 and 0[9 mM\ respectively[

O O HO OH O O

Me O OMe O O MeO OMe MeO MeO OH MeO OMe MeO O MeO O

MeO OMe MeO MeO HO OH O O OH OH (224a) (Ð)-Gomisin-J (224b) (+)-Gomisin-J (225) Gomisin-D (226) Gomisin-E

O MeO HO OH Br CH2 O MeO MeO

MeO MeO MeO HO CH2 MeO MeO MeO

MeO MeO MeO Br O MeO MeO HO O (227) Gomisin-N (228) Deoxyschizandrin (229) (230) Anolignan A

O O O O

H O MeO H MeO RO RO MeO MeO R = H H OH H O O O O (231) Interiotherin A (232) Schisantherin D

0[14[03[4 Miscellaneous Health Bene_ts] Anti!in~ammatory\ Antiasthmatic\ and Antidepressant Effects Kadsurenone "122#\ from P[ futokadsura "Piperaceae#\ is a platelet!activating factor\ a potent mediator of in~ammation\ and an asthma antagonist[200 Such e}ects have also been ascribed to fargesin "190#:eudesmin "72# from Ma`nolia biondii "Magnoliaceae#\201 as well as yangambin A "74# from Ocotea duckei "Lauraceae#202 and neojusticin A "123#\ justicidin B "088#\ taiwanin E "124#\ and its methyl ether "125# from Justicia procumbens "Acanthaceae#[203 Potential antiasthmatic agents are evaluated on their abilities to inhibit cyclic nucleotide phos! phodiesterase\ this being responsible for hydrolyzing cAMP and cGMP into their respective 4?! mononucleotides[ That is\ increases in cellular levels of cAMP and cGMP have been implicated in relaxation of the airway smooth muscle\ given that elevated levels of cAMP prevent the activation of pro!in~ammatory cells[ Lignans and norlignans with demonstrable cAMP phosphodiesterase Li`nans] Biosynthesis and Function 694

OMe O OR O O OMe O O O O MeO O O O MeO O O O O (233) Kadsurenone (234) Neojusticin A (235) R = H, Taiwanin E (236) R = Me, Taiwanin E methyl ether

inhibitory properties include] "¦#!pinoresinol "2a#:"−#!matairesinol "10a# from Forsythia species\204 cis!hinokiresinol "126#:oxy!cis!hinokiresinol "127# from Anemarrhena asphodeloides\205 and "¦#!syringaresinol!di!O!b!D!glucopyranoside "128#:"¦#!hydroxypinoresinol 3?\3ý!di!O!b!D!gluco! pyranoside "139# from Eucommia ulmoides bark[206 Additionally\ arylnaphthalene analogues have been synthesized and tested as cyclic nucleotide phosphodiesterase IV inhibitors] compound "130# inhibits phosphodiesterase "IC49 9[946 mM# and displays antispasmodic activities^ it is 7!fold more −0 −0 active than rolipram "ED49 1[2 mg kg versus 08 mg kg i[v[# in the guinea pig antigen!induced bronchoconstriction model[ However\ it was 7!fold less active than rolipran in a histamine!induced −0 −0 207 bronchospasmotic assay "ED49 9[97 mg kg versus 9[90 mg kg i[v[#[

OH OH

HO HO OH

(237) cis-Hinokiresinol (238) Oxy-cis-hinokiresinol

OMe OGlc OGlc OH O R1 O OMe OH OMe OH H O MeO O N O GlcO 2 GlcO R OMe OMe (239) (+)-Syringaresinol (240) (+)-Hydroxypinoresinol (241) 1 di-O-β-D-glucopyranoside 4,4'-di-O-β-D-glucopyranoside R = 6,7-(OEt)2 R2 = N-(2-methoxyethyl)

Magnoshinin "131# and magnosalin "132#\ isolated from Ma`nolia salicifolia buds\ also display anti!in~ammatory e}ects comparable to hydrocortisone acetate[208\219 Diphyllin acetyl apioside "133# and tuberculatin "134# are active against in~ammation induced by 01!O!tetradecanoylphorbol acetate "TPA#] the 49) inhibitory doses for acute TPA in~ammation were 9[16 and 0[12 mmol:ear for "133# and "134#\ respectively^ the former is a more potent inhibitor than indomethacin[210 Lignans also exhibit antidepressant activities\ e[g[\ prostalidins A\ B\ and C "135#Ð"137#\ from Justicia prostata "Acanthaceae#\ a plant native to the Western Himalayas[211 Finally\ the cardiovascular e}ects of lignans are very signi_cant] Siberian ginseng "Acanthopomax senticosus#\ which is widely used in Asia\ has the e}ect of helping sustain cardiovascular activity during prolonged exercise^212 this has been attributed to the lignan\ "¦#!syringaresinol di!O!b!D! glucoside "128#[213 695 Li`nans] Biosynthesis and Function

OR HO

OMe OH O O MeO MeO MeO O MeO OMe OMe MeO O MeO MeO MeO OMe OMe MeO O O (242) Magnoshinin (243) Magnosalin (244) R = Ac, Diphyllin acetyl apioside (245) R = H, Tuberculatin

2 R O O O O R1O

O O (246) R1 = H, R2 = OMe, Prostalidin A (247) R1 = Me, R2 = OMe, Prostalidin B (248) R1 = R2 = H, Prostalidin C

0[14[04 CONCLUDING REMARKS The foregoing discourse has described the knowledge gained in delineating how stereoselective and regiospeci_c control of coupling occurs in planta[ The discovery of dirigent protein mediated phenolic coupling\ leading to both "¦#! and "−#!pinoresinols "2a# and "2b#\ depending upon the species\ strongly suggests the involvement of related proteins which stipulate distinct coupling modes[ Indeed\ all research studies reveal that steps associated with both coupling and subsequent metabolic conversions are\ as for all other natural products\ under full biochemical control[ Re!examination of various claims for {{abnormal|| lignins\ {{secondary|| lignins and related sub! stances has revealed that they are nonlignin\ nonstructural\ oligomeric lignan infusions being secreted into neighboring preligni_ed cells "such as in heartwood# via specialized cells[ Additionally\ because of their susceptibility to further oxidation during isolation\ it cannot be ruled out that some of the sesquilignans are not\ in fact\ artifacts of the isolation process[ Additional work will lead to further clari_cation of the "oligomeric# lignan and lignin forming processes\ which must now be viewed as being fully distinct\ in terms of biochemical processes involved in their formation\ and their structural con_gurations\ as well as in their temporal and spatial deposition in vivo[ Finally\ the importance of the various lignan skeleta in both plant physiology "particularly defense# and in human nutrition and medicine continues to grow\ as the properties of this massive class of natural products continue to be discovered[

ACKNOWLEDGMENTS The authors thank the United States Department of Energy "DE!FG9286ER19148#\ the National Science Foundation "MCB98520879#\ the National Aeronautics and Space Administration Li`nans] Biosynthesis and Function 696 "NAG099053#\ the United States Department of Agriculture "8592511#\ McIntire!Stennis\ the Arthur M[ and Kate Eisig Tode Foundation\ and the Lewis B[ and Dorothy Cullman and G[ Thomas Hargrove Center for Land Plant Adaptation Studies for generous support of this study[

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L[ B[ Davin\ and N[ G[ Lewis\ 0888\ submitted for publication[ 007[ N[ G[ Lewis\ M[ A[ Costa\ L[ B[ Davin\ and Z[!Q[ Xia\ Patent Application] {{Recombinant Secoisolariciresinol Dehydrogenase\ and Methods of Use\|| 0887\ p[ 39[ 008[ H[ Adlercreutz\ K[ HoÃckerstedt\ C[ Bannwart\ E[ HaÃmaÃlaÃinen\ T[ Fotsis\ and S[ Bloigu\ Pro`ress in Cancer] Research and Therapy\ 0877\ 24\ 398[ 019[ H[ Adlercreutz\ in {{Natural Antioxidants and Food Quality in Atherosclerosis and Cancer Prevention\|| eds[ J[ T[ Kumpulainen and J[ K[ Salonen\ Royal Society of Chemistry\ Cambridge\ 0885\ p[ 238[ 010[ J[ E[ Bakke and H[ J[ Klosterman\ Proc[ No[ Dakota Acad[ Sci[\ 0845\ 09\ 07[ 011[ O[ Goldschmid and H[ L[ Hergert\ Tappi\ 0850\ 33\ 747[ 012[ L[ B[ Davin\ D[ R[ Gang\ M[ Fujita\ A[ M[ Anterola\ and N[ G[ Lewis\ in {{Proc[ 8th Internat[ Symp[ Wood Pulp[ Chem[\|| 0886\ p[ H2[ 013[ M[ Fujita\ D[ R[ Gang\ L[ B[ Davin\ and N[ G[ Lewis\ J[ Biol[ Chem[\ 0888\ 163[ 014[ T[ Umezawa and M[ Shimada\ Biosci[ Biotech[ Biochem[\ 0885\ 59\ 625[ 015[ D[ E[ Jackson and P[ M[ Dewick\ Phytochemistry\ 0873\ 12\ 0036[ 016[ W[ van Uden\ N[ Pras\ and H[ J[ Woerdenbag\ in {{Biotechnology in Agriculture and Forestry\|| ed[ Y[ P[ S[ Bajaj\ Springer!Verlag\ Berlin\ 0883\ vol[ 15\ p[ 108[ 017[ H[ J[ Wichers\ G[ G[ Versluis!De Haan\ J[ W[ Marsman\ and M[ P[ Harkes\ Phytochemistry\ 0880\ 29\ 2590[ 018[ W[ van Uden\ N[ Pras\ J[ F[ Visser\ and T[ M[ Malingre\ Plant Cell Rep[\ 0878\ 7\ 054[ 029[ W[ van Uden\ N[ Pras\ and T[ M[ Malingre\ Plant Cell\ Tiss[ Or`[ Cult[\ 0889\ 12\ 106[ 020[ W[ van Uden\ N[ Pras\ E[ M[ Vossebeld\ J[ N[ M[ Mol\ and T[ M[ Malingre\ Plant Cell\ Tiss[ Or`[ Cult[\ 0889\ 19\ 70[ 021[ W[ van Uden\ Pharm[ World[ Sci[\ 0882\ 04\ 30[ 022[ A[ J[ Broomhead\ M[ M[ A[ Rahman\ P[ M[ Dewick\ D[ E[ Jackson\ and J[ A[ Lucas\ Phytochemistry\ 0880\ 29\ 0378[ 023[ Z[!Q[ Xia\ L[ B[ Davin\ and N[ G[ Lewis\ Phytochemistry\ 0888\ submitted for publication[ 024[ M[ M[ Rao and D[ Lavie\ Tetrahedron\ 0863\ 29\ 2298[ 025[ M[ Kobayashi and Y[ Ohta\ Phytochemistry\ 0872\ 11\ 0146[ 026[ R[ Valsaraj\ P[ Pushpangadan\ U[ W[ Smitt\ A[ Adsersen\ S[ B[ Christensen\ A[ Sittie\ U[ Nyman\ C[ Nielsen\ and C[ E[ Olsen\ J[ Nat[ Prod[\ 0886\ 59\ 628[ 027[ T[ Deyama\ T[ Ikawa\ and S[ Nishibe\ Chem[ Pharm[ Bull[\ 0874\ 22\ 2540[ 028[ M[ A[ Bernards\ M[ L[ Lopez\ J[ Zajicek\ and N[ G[ Lewis\ J[ Biol[ Chem[\ 0884\ 169\ 6271[ 039[ M[ A[ Bernards and N[ G[ Lewis\ Polyphenols Actualites\ 0885\ 03\3[ 030[ M[ A[ Bernards and N[ G[ Lewis\ Phytochemistry\ 0887\ 36\ 804[ 031[ M[ H[ Chaves and N[ F[ Roque\ Phytochemistry\ 0886\ 35\ 768[ 032[ L[ Lajide\ P[ Escoubas\ and J[ Mizutani\ Phytochemistry\ 0884\ 39\ 0094[ 033[ I[ Sakakibara\ T[ Katsuhara\ Y[ Ikeya\ K[ Hayashi\ and H[ Mitsuhashi\ Phytochemistry\ 0880\ 29\ 2902[ 034[ I[ Sakakibara\ Y[ Ikeya\ K[ Hayashi\ and H[ Mitsuhashi\ Phytochemistry\ 0881\ 20\ 2108[ 035[ I[ Sakakibara\ Y[ Ikeya\ K[ Hayashi\ M[ Okada\ and M[ Maruno\ Phytochemistry\ 0884\ 27\ 0992[ 036[ T[ Yoshihara\ 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290[ 103[ P[ Rudman\ Holzforschun`\ 0854\ 08\ 46[ 104[ Atta!ur!Rahman\ M[ Ashraf\ M[ I[ Choudhary\ Habib!ur!Rehman\ and M[ H[ Kazmi\ Phytochemistry\ 0884\ 39\ 316[ 105[ M[ Takasugi and N[ Katui\ Phytochemistry\ 0875\ 14\ 1640[ 106[ A[ M[ Clark\ F[ S[ El!Feraly\ and W[!S[ Li\ J[ Pharm[ Sci[\ 0870\ 69\ 840[ 107[ H[ Kasahara\ M[ Miyazawa\ and H[ Kameoka\ Phytochemistry\ 0886\ 33\ 0368[ 108[ M[ Miyazawa\ H[ Kasahara\ and H[ Kameoka\ Phytochemistry\ 0882\ 23\ 0490[ 119[ M[ Miyazawa\ H[ Kasahara\ and H[ Kameoka\ Phytochemistry\ 0883\ 24\ 0080[ 110[ H[ Kasahara\ M[ Miyazawa\ and H[ Kameoka\ Phytochemistry\ 0885\ 32\ 000[ 111[ H[ Kasahara\ M[ Miyazawa\ and H[ Kameoka\ Nat[ Prod[ Lett[\ 0886\ 8\ 166[ 112[ M[ Hattori\ S[ Hada\ A[ Watahiki\ H[ Ihara\ Y[!Z[ Shu\ N[ Kakiuchi\ T[ Mizuno\ and T[ Namba\ Chem[ Pharm[ Bull[\ 0875\ 23\ 2774[ 113[ S[ Yamauchi\ F[ Ishibashi\ and E[ Taniguchi\ Biosci[ Biotech[ Biochem[\ 0881\ 45\ 0659[ 114[ S[ Yamauchi and E[ Taniguchi\ Biosci[ Biotech[ Biochem[\ 0881\ 45\ 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124[ T[ Yoshihara\ K[ Yamaguchi\ and S[ Sakamura\ A`ric[ Biol[ Chem[\ 0871\ 35\ 742[ 125[ J[ Harmatha and J[ Nawrot\ Biochem[ Syst[ Ecol[\ 0873\ 01\ 84[ 126[ K[ Matsui and K[ Munakata\ Tetrahedron Lett[\ 0864\ 13\ 0894[ 127[ K[ Matsui\ K[ Wada\ and K[ Munakata\ A`ric[ Biol[ Chem[\ 0865\ 39\ 0934[ 128[ K[ Munakata\ S[ Marumo\ K[ Ohta\ and Y[!L[ Chen\ Tetrahedron Lett[\ 0854\ 36\ 3056[ 139[ S[ D[ Elakovich and K[ L[ Stevens\ J[ Chem[ Ecol[\ 0874\ 00\ 16[ 130[ D[ Lavie\ E[ C[ Levy\ A[ Cohen\ M[ Evenari\ and M[ Y[ Guttermann\ Nature\ 0863\ 138\ 277[ 131[ M[ Szabo and A[ Garay\ Acta Botanica Academiae Scientiarum Hunàricae\ 0869\ 05\ 196[ 132[ P[ V[ Bhiravamurty\ R[ Das Kanakala\ E[ Venkata Rao\ and K[ V[ Sastry\ Curr[ Sci[\ 0868\ 37\ 838[ 133[ M[ M[ Chattaway\ Aust[ J[ Sci[ Res[ B\ 0838\ 1\ 116[ 134[ M[ M[ Chattaway\ Aust[ For[\ 0841\ 05\ 14[ 135[ R[ L[ Krahmer\ R[ W[ Hemingway\ and W[ E[ Hillis\ Wood Sci[ Technol[\ 0869\ 3\ 011[ 136[ J[ Ralph\ J[ J[ MacKay\ R[ D[ Hat_eld\ D[ M[ O|Malley\ R[ W[ Whetten\ and R[ R[ Sedero}\ Science\ 0886\ 166\ 124[ 137[ K[ D[ R[ Setchell\ A[ M[ Lawson\ F[ L[ Mitchell\ H[ Adlercreutz\ D[ N[ Kirk\ and M[ Axelson\ Nature\ 0879\ 176\ 639[ 138[ S[ R[ Stitch\ J[ K[ Toumba\ M[ B[ Groen\ C[ W[ Funke\ J[ Leemhuis\ J[ Vink\ and G[ F[ Woods\ Nature\ 0879\ 176\ 627[ 149[ M[ Axelson\ J[ SjoÃvall\ B[ E[ Gustafsson\ and K[ D[ R[ Setchell\ Nature\ 0871\ 187\ 548[ 140[ H[ Adlercreutz\ in {{Nutrition\ Toxicity\ and Cancer\|| ed[ I[ R[ Rowland\ CRC Press\ Boca Raton\ FL\ 0880\ p[ 026[ 141[ H[ Adlercreutz\ T[ Fotsis\ J[ Lampe\ K[ WaÃhaÃlaÃ\T[MaÃkelaÃ\ G[ Brunow\ and T[ Hase\ Scand[ J[ Clin[ Lab[ Invest[\ 0882\ 42\4[ 142[ H[ Adlercreutz\ J[ van der Wildt\ J[ Kinzel\ H[ Attalla\ K[ WaÃhaÃlaÃ\T[MaÃkelaÃ\ T[ Hase\ and T[ Fotsis\ J[ Steroid Biochem[ Molec[ Biol[\ 0884\ 41\ 86[ 143[ M[ Axelson and K[ D[ R[ Setchell\ FEBS Lett[\ 0870\ 012\ 226[ 144[ K[ D[ R[ Setchell\ A[ M[ Lawson\ S[ P[ Borriello\ R[ Harkness\ H[ Gordon\ D[ M[ L[ Morgan\ D[ N[ Kirk\ H[ Adlercreutz\ L[ C[ Anderson\ and M[ Axelson\ Lancet\ 0870\ 3[ 145[ M[ S[ Kurzer\ J[ W[ Lampe\ M[ C[ Martini\ and H[ Adlercreutz\ Cancer Epidemiol[\ Biomarkers and Prev[\ 0884\ 3\ 242[ 146[ S[ I[ MaÃkelaÃ\ L[ H[ PylkkaÃnen\ R[ S[ S[ Santti\ and H[ Adlercreutz\ J[ Nutr[\ 0884\ 014\ 326[ 147[ M[ E[ Martin\ M[ Haourigui\ C[ Pelissero\ C[ Benassayag\ and E[ A[ Nunez\ Life Sci[\ 0885\ 47\ 318[ 148[ H[ Adlercreutz\ C[ Bannwart\ K[ WaÃhaÃlaÃ\T[MaÃkelaÃ\ G[ Brunow\ T[ Hase\ P[ J[ Arosemena\ J[ T[ Kellis\ Jr[\ and L[ E[ Vickery\ J[ Steroid Biochem[ Molec[ Biol[\ 0882\ 33\ 036[ 159[ C[ Wang\ T[ MaÃkelaÃ\ T[ Hase\ H[ Adlercreutz\ and M[ S[ Kurzer\ J[ Steroid Biochem[ Molec[ Biol[\ 0883\ 49\ 194[ 150[ L[ U[ Thompson\ M[ M[ Seidl\ S[ E[ Rickard\ L[ J[ Orcheson\ and H[ H[ S[ Fong\ Nutr[ Cancer\ 0885\ 15\ 048[ 151[ L[ U[ Thompson\ S[ E[ Rickard\ L[ J[ Orcheson\ and M[ M[ Seidl\ Carcinoènesis\ 0885\ 06\ 0262[ 152[ M[ Jenab and L[ U[ Thompson\ Carcinoènesis\ 0885\ 06\ 0232[ 153[ P[ I[ Musey\ H[ Adlercreutz\ K[ G[ Gould\ D[ C[ Collins\ T[ Fotsis\ C[ Bannwart\ T[ MaÃkelaÃ\K[WaÃhaÃlaÃ\ G[ Brunow\ and T[ Hase\ Life Sci[\ 0884\ 46\ 544[ 154[ H[ Adlercreutz\ Y[ Mousavi\ J[ Clark\ K[ HoÃckerstedt\ E[ HaÃmaÃlaÃinen\ K[ WaÃhaÃlaÃ\T[MaÃkelaÃ\ and T[ Hase\ J[ Steroid Biochem[ Molec[ Biol[\ 0881\ 30\ 220[ 155[ M[ SchoÃttner\ G[ Spiteller\ and D[ Gansser\ J[ Nat[ Prod[\ 0887\ 50\ 008[ 156[ H[ Adlercreutz\ H[ Honjo\ A[ Higashi\ T[ Fotsis\ E[ HaÃmaÃlaÃinen\ T[ Hasegawa\ and H[ Okada\ Am[ J[ Clin[ Nutr[\ 0880\ 43\ 0982[ 157[ S[ P[ Borriello\ K[ D[ R[ Setchell\ M[ Axelson\ and A[ M[ Lawson\ J[ Appl[ Bacteriol[\ 0874\ 47\ 26[ 158[ K[ D[ R[ Setchell\ A[ M[ Lawson\ S[ P[ Borriello\ H[ Adlercreutz\ and M[ Axelson\ Falk Symp[\ 0871\ 20 "Colonic Carcinog[#\ 82[ 169[ K[ Prasad\ Mol[ Cell[ Biochem[\ 0886\ 057\ 006[ 160[ K[ Yamashita\ Y[ Nohara\ K[ Katayama\ and M[ Namiki\ J[ Nutr[\ 0881\ 011\ 1339[ 161[ K[ Yamashita\ Y[ Iizuka\ T[ Imai\ and M[ Namiki\ Lipids\ 0884\ 29\ 0908[ 162[ M[ G[ Kelly and J[ 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